Index

Page references followed by f denote figures; those followed by t denote tables.

AAbazyme, LLC, 424Accuracy, ELISA, 641Acidic stripping buffer (recipe), 528Acids

disposal of, 821safe handling of, 820, 823

Activated carbon, coupling antigens to, 63Activation-induced deaminase (AID), 17Adaptive (acquired) immunity

clonal selection and, 4–6, 5fdescribed, 1–2link to innate immunity, 2–3

ADCC (antibody-dependent cellular cytotoxic-ity) by flow cytometry (proto-col), 751–754, 752f–753f

Adherent cellsgrowing

on coverslips or multiwell slides(protocol), 686

on tissue culture dishes (protocol), 687preparing for staining, 679

Adjuvants, 2, 33–34, 113–116advantages and disadvantages of specific,

114taluminum hydroxide, 116, 148–149Freund’s, 114–115, 142–143, 143fGERBU, 116Hunter’s TiterMax, 115, 145Magic Mouse, 115, 146–147overview of, 113–114Pam3Cys-Ser-(Lys)4, 115–116protocols, 136–149

aluminum hydroxide (alum) adjuvantpreparation, 148–149

Freund’s adjuvant preparation, 142–143,143f

Hunter’s TiterMax adjuvant use, 145Magic Mouse adjuvant use, 146–147Ribi adjuvant use, 144

Ribi, 115, 144as TLR agonists, 2

Adoptive transfer immunization of mice(protocol), 191

Affidity of antibody–antigen interaction, 24–28,25t, 26f–27f

bivalent bindingof antibodies to polymeric antigens, 25,

26fto antigens immobilized on solid

supports, 27f, 28factors affecting strength of binding, 25tintermolecular bridges and, 25, 26fmonoclonal antibody binding to multimeric

antigen, 25multivalent complexes, 25–28, 26f–27f

secondary reagents and, 27f, 28Affinity. See also Affinity purification

of antibody–antigen interaction, 24of IgG versus IgM, 39measurement of, 358–361

ELISA, 358–359equilibrium dialysis, 358labeling experiments, 359–360optical biosensor methods, 360–361surface plasmon resonance (SPR), 360

secondary response and, 33Affinity constants, 24, 358Affinity maturation, 18, 127Affinity purification, 377–382

chromatography for cleanup and purificationof antibody conjugates, 465

of modification state–specific antibodies,379–381, 379f–380f, 382f

overview, 377peptide affinity purification (protocol),

403–404of peptide-specific antibodies, 378of protein-specific antibodies, 378

Affinity tag, 55, 56t, 58, 539Agarose, coupling antigens to, 63AH selection medium (recipe), 351AID (activation-induced deaminase), 17Aldehyde cross-linking sites for labeling

antibody, 432, 433tAlignment algorithm, 51Alkaline phosphatase

in antibody capture assays, 210, 238–241antibody conjugation to, 448antigen detection on immunoblots, 519,

521–524detecting alkaline phosphatase-labeled cells

using BCIP-NBT, 719–720using NABP-NF, 718

SEAP (secreted embryonic alkalinephosphatase), 338, 339f

Alkaline phosphatase buffer (recipe), 523, 720Alkylation of proteins before running

on a gel, 765Allelic exclusion, 17–18Allophycocyanin, 448Aluminum hydroxide adjuvant, 113, 114t, 116

preparation protocol, 148–149Alzheimer’s disease, staining of amyloid plaques

associated with, 430fAmerican Type Culture Collection (ATCC), 309,

823Amido Black, 497Amines

cross-linking sites for labeling antibody,431–432, 432t, 433t

labeling antibodies with NHS-LC-Biotin(protocol), 436–437

Amino acids, 780tcodon usage, 783t–784tmobility of residues, 52substitutions, 786t

Aminoethylcarbazole, detecting horseradishperoxidase-labeled cells using(protocol), 717

Aminopterin, 205, 218, 316Amino-terminal regions, coupling to, 54Ammonium persulfate (APS), 491–492, 494Ammonium sulfate, saturated (recipe), 386Ammonium sulfate purification of antibodies,

372–373, 385–386Ammonium sulfate saturation tables, 778tAmphoteric detergents, 805, 806tAnalytical ultracentrifugation, 361Anaphylactic shock, 122Anergy, 38Anesthesia. See also specific anesthetic agents

choice of, 112, 113tdepth of, 112, 138protocols

for mice, rats, and hamsters, 136–138, 137ffor rabbits, 139–141

Animals. See also specific specieshumane treatment of, 821immunizing, 107–199killing mice, rats, and hamsters using carbon

dioxide asphyxiation, 194–195legal and moral considerations of use, 108–109

Anion-exchange chromatography, 373–374Anionic detergents, 806tAnnexin V, 749–750Antibiotics

addition to culture media, 304, 306t, 310,312, 325, 340–341

resistance by Mycoplasma, 312, 312t, 313ridding cell lines of contaminating

microorganisms by antibiotics(protocol), 325–326

ridding cells of mycoplasma contaminationusing antibiotics and single-cellcloning (protocol), 340–341

Antibodycharacterizing, 353–370

antibody backbone, 361–364effector domains, 362, 364isotype, 361–362, 363t

antigen binding, 353–361affinity, 358–361antibody specificity, 353–354antigenic epitopes, 354–357, 356fcomplementarity determining

regions (CDRs), 357–358isotype determination of rodent-derived

monoclonal antibodies using sand-wich ELISA (protocol), 366–370

825

Antibody (Continued)classes of

characteristics of, 13tsubclasses, 10switching, 18f, 19, 39, 233–234

cross-reactivity, 6defined, 2diversity, 14effector functions mediated by, 6–7, 7fengineering (see Engineering antibodies)labeling (see Labeling antibody)monoclonal (see Monoclonal antibodies)polyclonal (see Polyclonal antibodies)purification (see Antibody purification)recombinant (see Recombinant antibodies)responses, 31–40specificity, 23, 353–354storage, 383–384structure (see Antibody structure)synthetic, 235–236

Antibody–antigen complexaffinity, 358–361amount of complex formed, content of, 24formation in immunoprecipitation, 538structure of, 21–23, 22f

antigen binding site, 21–22, 22fconformational changes, 23epitopes, 22–23noncovalent interactions, 23specificity and, 23

Antibody–antigen interactions, 21–28affinity, 24avidity, 24–28, 25t, 26f–27fstructure of the antibody–antigen complex,

21–23, 22fAntibody binding

controls for, 681overview, 680–681

Antibody-binding assay (protocol), simplemultiplexed, 742–745, 744f, 744t

Antibody capturecapture or sandwich ELISA, 211fdirect ELISA, 211fdot blots, 213, 213findirect ELISA, 211foverview of, 209–213, 210f–211f, 212t,

213fprotocols

determining the class and subclass of amonoclonal antibody using anti-body capture on antigen-coatedplates, 294–296, 295f

determining the class and subclass of amonoclonal antibody using anti-body capture on anti-immuno-globulin antibody-coated plates,297–299

isotype determination of rodent-derivedmonoclonal antibodies usingsandwich ELISA, 366–370

on nitrocellulose membranedot blot, 242–243high-throughput western blot assay

for hybridoma screening,244–245

on permeabilized whole cellsimmunofluorescence, 253–254intracellular staining by flow

cytometry/FACS, 249–250in polyvinyl chloride wells

enzyme-linked detection (indirectELISA), 238–239

enzyme-linked detection whenimmunogen is an immunoglo-bulin fusion protein (indirectELISA to detect Ig fusionproteins), 240–241

on tissue sections (immunohistochemis-try), 255–257

on whole cellscell-surface binding (surface staining

by flow cytometry/FACS),246–248

cell-surface binding (surface stainingby immunofluorescence),251–252

Antibody-coated antigens, injecting, 64Antibody complex, avidity of, 24–28, 25t,

26f–27fAntibody conjugates, cleanup and purification

of, 464–468affinity chromatography, 465desalting or buffer exchange using

size-exclusion chromatography(protocol), 467–468

dialysis, 465–466ion-exchange chromatography, 464–465overview, 464–466, 464treversed-phase chromatography, 465size-exclusion chromatography, 464, 464t,

467–468Antibody-dependent cellular cytotoxicity

(ADCC) by flow cytometry(protocol), 751–754, 752f–753f

Antibody footprinting, 356Antibody-producing cells, types of, 3fAntibody purification, 371–404

affinity purification, 377–382of modification state–specific antibodies,

379–381, 379f–380f, 382foverview, 377of peptide-specific antibodies, 378of protein-specific antibodies, 378

large-scale antibody production, 382–383overview, 371–384

affinity purification, 377–382, 403–404anion-exchange chromatography,

373–374chicken (IgY) antibodies, 375–376,

397–400hydroxyapatite chromatography,

374–375IgM purification, 376–377immobilized metal affinity chromatog-

raphy (IMAC), 375ion-exchange methods, 374–375isolation of a total IgG fraction, 372–373

ammonium sulfate precipitation,372–373, 385–386

caprylic acid IgG isolation, 373,387–388

large-scale antibody production,382–383

Protein A chromatography, 375,393–394

Protein G chromatography, 375,395–396

purification of Fab and F(ab0)2fragments, 376

protocols, 385–404ammonium sulfate fractionation of

antibodies, 385–386conjugation of peptides to thiol-reactive

gel, 401–402

high-pH DEAE chromatography,391–392

isolation of IgY from chicken eggs:polyethylene glycol method,399–400

isolation of IgY from chicken eggs:sodium sulfate method, 397–398

low-pH DEAE chromatography,389–390

peptide affinity purification, 403–404preparation of antibody using caprylic

acid, 387–388Protein A purification, 393–394Protein G purification, 395–396

Antibody responses, 31–40adjuvants and, 33–34antigen presentation, 33–35, 35fantigen type and, 33–34B-cell activation, 33–34, 34f, 36–37, 37f, 38tB cell differentiation and, 39–40, 40fclass II proteins and, 34–36germinal centers and, 39immune tolerance, 37–39memory cells, 32–33primary response, 32secondary response, 32, 33T-cell receptors and, 35–36, 37fT follicular helper cell activation, 36, 37f

Antibody sandwich ELISAimmunometric (protocol), 651–657, 652f,

654t, 655f–656fimmunometric double-antibody (protocol),

658–660, 658fAntibody structure

antigen-binding sites, 10–11, 11f, 12, 14classes of antibodies and, 13tconstant regions, 12–13diversity of, 14Fab fragments, 10, 11f, 14Fc fragments, 10–11, 11fheavy chains, 10–14, 12f–13flight chains, 10, 12, 12f–13f, 14variable regions, 12–14

Antigencoupling (see Coupling antigens)

Antigen(s). See also Immunogen(s)binding to class II proteins, 35–36bivalent binding to antigens immobilized on

solid supports, 27f, 28detection on immunoblots, 519–525

enzyme-based, 519, 521–524with fluorochromes, 519–520, 525

modification, 61–64nonresponders to, 36phagocytosis and presentation, 33, 34–35,

35fpreparation using baculovirus expression

system (protocol), 101–103processing, 34selection (see Antigen selection)T-cell-dependent, 33T-cell-independent, 34

Antigen–antibody complexaffinity, 358–361amount of complex formed, content of, 24formation in immunoprecipitation, 538structure of, 21–23, 22f

antigen binding site, 21–22, 22fconformational changes, 23epitopes, 22–23noncovalent interactions, 23specificity and, 23

826 / Index

Antigen binding, 353–361affinity, 358–361antibody specificity, 353–354antigenic epitopes, 354–357, 356fcomplementarity determining regions

(CDRs), 357–358Antigen-binding sites

diversity of, 17structure of, 10–11, 11f, 12, 14, 21–22, 22f

Antigen captureoverview of, 210f, 213–214protocols

on nitrocellulose membrane: reverse dotblot, 261–262

in solution: immunoprecipitation, 263in 96-well plates (capture or sandwich

ELISA), 258–260Antigen dialysis buffer (recipe), 80Antigen electrophoresis buffer (recipe), 80Antigenicity

defined, 2predicting, 51–52

Antigenicity prediction software, 51Antigen modification, 61–64

coupling antigens, 62–64to beads, 63bifunctional cross-linkers, 50thaptens, 50immune complexes as antigens, 63–64to protein carriers, 62to red blood cells, 63with T-cell receptor–MHC-class II

protein-binding sites, 62–63protocols

arsynyl coupling, 67–68denaturation, 69dinitrophenol coupling, 66

Antigen-presenting cells (APCs), 2, 33–35, 35fT follicular helper cell activation, 36, 37ftransfected dendritic cell immunizations

(protocol), 104–106Antigen selection, 43–106

antigen modification, 61–64immunogenicity, 44–49

failure of animal to respond, 46–47polyclonal versus monoclonal antibodies,

47–48, 47tproperties of good immunogens, 45–46,

46tselecting the antigen and, 48, 49tsynthetic class II–T-cell receptor sites,

47tprotocols, 66–106

antigen preparation using baculovirusexpression system, 101–103

autoradiography, 83–84Coomassie Brilliant Blue staining, 73copper chloride staining, 75coupling antigens to red blood cells, 72cross-linking peptides to KLH with

maleimide, 85–86electroelution of protein antigens from

polyacrylamide gel slices, 79–80electrophorectic transfer to nitrocellulose

membranes, 81–82fragmenting a wet gel slice, 77GST-fusion protein preparation from

bacteria, 87–88His-fusion protein preparation from

bacteria, 89–91immune complex preparation for

injection, 70–71

live cell preparation for immunization,100

lyophilization of gel slice, 78MBP-fusion protein preparation from

bacteria, 92–94mFc- and hFc-fusion protein preparation

from mammalian cells, 97–99modifying antigens by arsynyl coupling,

67–68modifying antigens by denaturation, 69modifying antigens by dinitrophenol

coupling, 66sarkosyl preparation of antigens from

bacterial inclusion bodies, 95–96side-strip method, 76sodium acetate staining, 74transfected dendritic cell immunizations,

104–106questions to ask, 44sources of immunogens, 49–61, 49t

cDNA, 49t, 61cell and tissue lysates, 49t, 59–60haptens, 50purified native proteins (pure antigens),

49t, 54–55recombinant proteins, 49t, 55–59

advantages and disadvantages of use,49t

from bacterial overexpression vectors,55–56, 56t

from baculovirus overexpressionvectors, 56

choosing between proteins andpeptides, 57–59, 57t, 58t

from mammalian overexpressionvectors, 56–57

synthetic peptides, 49t, 50–54advantages and disadvantages of use,

49t, 51carrier selection, 54choosing over recombinant protein,

57–59coupling strategy, 53–54designing the peptide, 51–52peptide design against highly

homologous extracellularproteins, 53

peptide sequence choice, 52size of peptide, 53

Anti-immunoglobulin antibodies, avidity and,27f, 28

APCs. See Antigen-presenting cellsApoptosis assessment (protocol), 749–750, 750fAPS (ammonium persulfate), 491–492, 494Arsynyl coupling, modifying antigens by

(protocol), 67–68Ascites

inductionin BALB/c mice using myeloma cells

(protocol), 183induction and collection (protocol),

344–345in mice, 129using Freund’s adjuvant (protocol), 182

ridding cell lines of contaminatingmicroorganisms by passagethrough mice (protocol),328–329

ridding cells of mycoplasma contaminationby passage through mice(protocol), 342–343

serial passage of tumors in mice, 345

storing tissue culture supernatants andascites (protocol), 347–348

Asphyxiation, killing mice, rats, and hamstersusing carbon dioxide (protocol),194–195

ATCC (American Type Culture Collection), 309,823

Atipamezole for anesthesia, 113tAttaching suspension cells to slides (protocols)

using cytocentrifuge, 688using poly-L-lysine, 689

Autoclave safety, 820Autoradiography, 55, 775

protocol, 83–84Autoreactivity, 6Avertin for anesthesia, 113tAvidin, 6328-azaguanine selection, 205, 316–317, 349Azaserine, 205, 218, 316Azaserine/hypoxanthine (AH) selection

medium, 316, 350–351

BBack-fusing, 229Backscatter interferometry, 361Bacteria

biological safety procedures, 822–823contamination of cell cultures, 230f, 277

overview, 309–310ridding cell lines of by antibiotics

(protocol), 325–326ridding cell lines of by passage through

mice (protocol), 328–329ridding cell lines of with peritoneal

macrophages (protocol), 327fusion protein preparation protocols

GST-fusion proteins, 87–88His-fusion proteins, 89–91MBP-fusion proteins, 92–94

sarkosyl preparation of antigens frombacterial inclusion bodies(protocol), 95–96

Bacterial expression, 809–818maps

b-gal fusion vectors, 815fgateway expression vectors, 817flgt11, 812fpcDNA 3.1, 816fpDEST 8, 818fpDEST 15, 817fpDEST 27, 817fpET-SUMO, 816fpFastBac1, 816fplasmids, 813f–818fTrp fusion vectors, 814fT7 vectors, 813f

screening forchloroform lysis, 811SDS lysis, 810

systems, 55–56, 57t, 58, 58tBacterial overexpression vectors, preparing

antigens from, 55–56, 56t, 57tBaculovirus expression system, 57t, 58–59, 58t

antigen preparation using (protocol),101–103

materials, 101protein expression and purification, 103transfection of Sf9 cells and virus stock

generation, 101–102viral titer and plaque assay, 102–103virus stock harvest and amplification, 102

Index / 827

Baculovirus overexpression vectors, preparingantigens from, 56, 57t

BAFF, 5, 31–32, 32f, 34, 34f, 38BAFF receptor (BAFF-R), 32, 32f, 38Bases

disposal of, 821safe handling of, 820, 823

B-cell activating factor of the TNF family(BAFF), 5, 31–32, 32f, 34, 34f, 38

B-cell activation, signals for, 33–34, 34f, 36–37,37f, 38t

B-cell receptor (BCR), 1, 2f, 4–5cross-linking, 36, 37fsignaling and B-cell survival, 31–32, 32f

B cellsactivation of, 1, 3described, 1–2differentiation, 3–4, 39–40, 40f, 45memory B cells, 3, 3f, 4, 32–33, 39–40, 40fplasma cells, 32, 39–40, 40fsurvival, signaling and, 31–32, 32fup-regulation of cytokine receptors, 37

B-cell tolerance, 6, 38, 46B-cell tumors, 202BCIP-NBT, detecting alkaline phosphatase-

labeled cells using (protocol),719–720

BCIP stock solution for AP (recipe), 523BCR. See B-cell receptorBeadBeater Cell Disruptor, 580, 582Beads

bead-based enzyme-linked immunosorbantassay (ELISA), 642–643

coupling antigens to, 63glass beads, lysing yeast cells with (protocol),

579–581using lysis buffer 2 without detergents

(protocol), 582–584magnetic beads-immunoprecipitation,

540–541, 793Bentonite, coupling antigens to, 63Benzaldehyde, 377Berdoz primer set, 424b-galactosidase detection in

b-galactosidase-labeled cellsusing X-gal (protocol), 721

b-gal fusion vectors, 815fb-mercaptoethanol, 474b-turns, 52Biacore AB, 360Bicinchoninic acid protein quantification, 792Bifunctional cross-linkers, 50t, 62Binding antibodies (protocols)

to attached cells or tissues, 709–710to cells in suspension, 711–712

Binding competition assays, 355, 356fBiolayer interferometry, 360Biological safety procedures, 822–823Bio-Rad (DEAE Affi-Gel Blue), 374–375Biotin, 681

in antibody capture assays, 210labeling antibodies by biotinylation, 435–441

labeling using biotin polyethylene oxide(PEO) iodoacetamide (protocol),438–439

labeling using Hydrazide-LC-biotin(protocol), 440–441

labeling with NHS-LC-Biotin (protocol),436–437

size of, 435Bivalent binding

of antibodies to polymeric antigens, 25, 26f

to antigens immobilized on a solid phase,27f, 28

BLAST analysis, 51Blocking and incubation of immunoblots with

antibodies, 509–518immunoblots prepared with

immunoprecipitated proteinantigens (immunoprecipitation/western blotting), 515–518

immunoblots prepared with whole-celllysates and purified proteins(straight western blotting),510–514

overview, 509Blocking buffer (recipe), 370Blocking buffers, ELISA, 635–636

BLOTTO, 635bovine serum albumin (BSA)-based, 635casein-based, 636gelatin, 636protein-free, 636serum, 636

Blocking/neutralizing or activating antibodies,screening for, 215

Blocking nonspecific binding sites on themembrane for immunoblotting,475–476, 476t

Blocking reagents used for immunoblotting,476–477

Blocking solutions (recipe), 513, 517, 529Blood sampling

methods of, 128ttest bleeds, 127–129

BLOTTO, 253–254, 509, 635B lymphocytes. See B cellsBM Cyclin Mycoplasma Removal Kit, 341Boehringer Mannheim BM Cyclin Mycoplasma

Removal Kit, 341Bolton-Hunter reagent, 461Boosts

final before fusion, 218–220preparing myeloma cells for fusion, 219,

219fpreparing partner cells for fusion, 218–219preparing splenocytes for fusion,

219–220, 219f–221fresponse to, 124timing of, 124

Borate buffer (pH 8.0, 1�) (recipe), 595Borate buffer (pH 9.0, 1�) (recipe), 595Bordetella pertussis, 113, 116, 148–149Bouin’s fixative (recipe), 699Bovine serum albumin (BSA)

based blocking buffers, 509, 635choosing as carrier, 54coupling antigens to, 62

Bradford protein quantification, 789spot test, 790

Bromochloroindoyl phosphate/nitro blue tetra-zolium (BCIP-NBT), detectingalkaline phosphatase-labeled cellsusing (protocol), 719–720

BSA. See Bovine serum albuminBuffer A for cross-linking antibodies to beads

(recipe), 595, 599Buffer A for dounce homogenization (recipe), 573Buffer B for dounce homogenization (recipe), 573Buffer C for dounce homogenization (recipe), 574Buffer D for dounce homogenization (recipe), 574Buffer exchange using size-exclusion chroma-

tography (protocol), 467–468Buffers, commonly used, 800t

CCalcein AM, 751, 752f–753f, 754Caprylic acid, preparation of antibody using,

373, 387–388Capture ELISA. See also Sandwich ELISA

antibody capture assay, 211fantigen capture assay, 214antigen capture in 96-well plates (protocol),

258–260Carbohydrates

biotinylating antibodies usinghydrazide-LC-biotin (protocol),440–441

cross-linking sites for labeling antibody, 432,433t

immunogen form for immunizing animals,117t, 118t, 119

Carbonate buffer (recipe), 650, 656, 660, 668, 673Carbon dioxide

for anesthesia, 112killing mice, rats, and hamsters using carbon

dioxide asphyxiation (protocol),194–195

Carbonyl cross-linking sites for labeling anti-body, 431–432, 432t, 433t

Carboxy-terminal sequencesas antipeptide targets, 52coupling to, 54

Carrierschoosing appropriate, 54coupling antigens to protein carriers, 62cross-linking peptides to KLH with

maleimide (protocol), 85–86Casein-based blocking buffers, 636Cationic detergents, 806tCats

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgG binding affinity for Proteins A, G, and L,540t

CBA (Cytometric Bead Assay), 232, 300CD4, 35CD40, 34CD40-CD40 L interaction, 37, 37fcDNA

electroporation into E. coli, 418–419immunization, 125–126

in mice, rats, and hamsters (protocol),192–193, 193f

as immunogen source, 49t, 61library construction, 417–420preparation

in creation of recombinant antibodiesusing degenerate oligionucleo-tides (protocol), 407–409

in creation of recombinant antibodiesusing phage display (protocol),417–420

in creation of recombinant antibodiesusing 50-RACE (protocol),410–411

cDNA synthesis buffer (recipe), 411CDRs. See Complementarity determining

regionsCell-based ELISA, direct and indirect (protocol),

661–665, 663f–664fCell cultures, 304–309

antibiotics for, 304, 306t, 310, 312, 325,340–341

checking cell lines for HPRT deficiency,350–352

contamination of, 309–313

828 / Index

by bacteria, fungi, or yeast, 309–310,325–329

by mycoplasma, 310–313, 311t–312t,330–343

ridding cells of (protocols), 325–329,340–343

testing for (protocols), 330–339counting myeloma or hybridoma cells

(protocol), 318–319, 319fdetergent lysis of tissue culture cells

(protocol), 565–567drug selection, 316–317, 349freezing cells for liquid nitrogen storage

(protocol), 321–322media, 304–307, 305t–306trecovering cells from liquid nitrogen storage

(protocol), 323–324sending and receiving hybridomas and

myelomas, 309storage of, 309, 321–324supernatant collection, 314–315, 346supernatant storage, 347suppliers of media, 307ttechnique for, 307–308, 308fviability checks (protocol), 320

Cell lysis for immunoprecipitationdenaturing lysis (protocol), 588–590detergent lysis of animal tissues (protocol),

568–570detergent lysis of tissue culture cells

(protocol), 565–567differential detergent lysis of cellular

fractions (protocol), 575–578freezing cell pellets for large-scale

immunoprecipitation (protocol),563–564

lysing yeast cells using a coffee grinder(protocol), 585–587

lysing yeast cells with glass beads (protocol),579–581

using lysis buffer 2 without detergents(protocol), 582–584

lysis using dounce homogenization(protocol), 571–574

overview, 534–537lysis buffers, 534–535lysis of tissue, 536–537lysis of tissue culture cells, 535–536lysis of yeast, 537protease inhibitor use, 535, 536t

protocols, 565–590Cell-permanent dye, 742–743Cell repositories, 309Cells

cultures of (see Cell cultures)as immunogen source, 49t, 59–60mouse antibody production (MAP) tested,

60staining (see Cell staining)

Cell smearpreparation (protocol), 690preparing from tissue samples or cell cultures

(protocol), 700Cell staining, 675–733

antibody choice for, 677–678, 677tmonoclonal antibodies, 677t, 678polyclonal antibodies, 677–678, 677t

major constraints, 676overview, 676protocols, 686–733

attaching suspension cells to slidesusing cytocentrifuge, 688

using poly-L-lysine, 689attaching yeast cells to slides using

poly-L-lysine, 691–692binding antibodies

to attached cells or tissues, 709–710to cells in suspension, 711–712

cell smear preparation, 690counterstaining, 729detecting alkaline phosphatase-labeled

cellsusing BCIP-NBT, 719–720using NABP-NF, 718

detecting b-galactosidase-labeled cellsusing X-gal, 721

detecting fluorochrome-labeled reagents,722–724

detecting gold-labeled reagents, 725–726detecting horseradish peroxidase-labeled

cellsusing aminoethylcarbazole, 717using chloronaphthol, 716using diaminobenzidine, 713–714,

714fusing diaminobenzidine and metal

salts, 715detecting iodine-labeled reagents,

727–728embedding cultured cells in matrigel,

701–702fixing attached cells

in organic solvents, 703–704in paraformaldehyde or glutaralde-

hyde, 705–706fixing suspension cells with paraformal-

dehyde, 707general overview, 679–683

antibody binding, 680–681detection, 681–683, 682t, 683tfixation, 680preparation of cells and tissues,

679–680growing adherent cells

on coverslips or multiwell slides, 686on tissue culture dishes, 687

heat-induced epitope retrieval, 698–699lysing yeast, 708mounting cell or tissue samples

in DPX, 730in Gelvatol or Mowiol, 731

photographing samples, 733preparing cell smears from tissue samples

or cell cultures, 700preparing frozen tissue sections,

693–695preparing paraffin tissue sections,

696–697strength of antibody binding, 25ttroubleshooting background problems,

683–684Cell-surface binding

surface staining by flow cytometry/FACS,246–248

surface staining by immunofluorescence,251–252

Cell surface receptor internalization, 216fChasing medium (recipe), 556Chemically defined medium, 304, 307tChemicals, properties of common hazardous,

823–824Chemifluorescent substrates, ELISA, 638–639Chemiluminescence

antigen detection on immunoblots, 522

reprobing membranes after immunoblotting,526

substrates, ELISA, 638Chickens

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgY antibodies, 375–376, 397–400isolation of IgY from chicken eggs:

polyethylene glycol method(protocol), 399–400

isolation of IgY from chicken eggs:sodium sulfate method(protocol), 397–398

ChIP (chromatin immunoprecipitation). SeeImmunoprecipitation, chromatin

Chloroauric acid, 455, 459–460Chloroform lysis, screening for bacterial

expression by, 811Chloronaphthol, detecting horseradish

peroxidase-labeled cells using(protocol), 716

Chromatin immunoprecipitation (ChIP). SeeImmunoprecipitation, chromatin

Chromatography. See specific chromatographytypes

Chromogenic antigen detection on immuno-blots, 477, 522

Chromogenic substratesELISA, 637–638yielding water-insoluble products, 797tyielding water-soluble products, 797t

Chromosome loss in hybridomas, 205Cibacron Blue F3GA, 375Cimetidine, 116Citraconic anhydride, 62Citrate binding buffer (recipe), 98Citrate elution buffer (recipe), 99Classes of antibodies

characteristics of, 13tsubclasses, 10switching, 18f, 19, 39, 233–234

Class II proteinantigen binding to, 35–36diversity of, 36structure, 34–35

Class switching, 18f, 19, 39, 233–234Cleanup and purification of antibody conjugates,

464–468affinity chromatography, 465desalting or buffer exchange using

size-exclusion chromatography(protocol), 467–468

dialysis, 465–466ion-exchange chromatography, 464–465overview, 464–466, 464treversed-phase chromatography, 465size-exclusion chromatography, 464, 464t,

467–468Clonal deletion, 5f, 6, 38Clonal dominance, 127Clonal selection, 4–6, 5fCloning

single-cell cloning by growth in soft agar,288–290

single-cell cloning by limiting dilution,286–287

cMyc epitope tag, 539Codon-optimized bacteria, 56Codon usage, 783t–784tCoffee grinder, lysing yeast cells using

(protocol), 585–587Coimmunoprecipitation, 532

Index / 829

Colloidal gold, 455, 459–460Colorimetric substrates. See Chromogenic

substratesCommercial liquids, concentrations of, 803tCompetition assays, for epitope binding, 355, 356fCompetitive ELISA

direct (protocol), 666–669, 666findirect (protocol), 670–673, 670f, 673f

Complementarity determining regions (CDRs),14, 17–18, 17f14, 22, 22f

described, 357sequencing, 357–358

Complete Freund’s adjuvant, 113, 114, 142Conjugation of peptides to thiol-reactive gel,

401–402Contamination

of cell cultures, 309–313by bacteria, fungi, or yeast, 309–310,

325–329by mycoplasma, 310–313, 311t–312t,

330–343ridding cells of (protocols), 325–329,

340–343testing for (protocols), 330–339

of a cloned line, 230–231in fusion wells, 229–230

Coomassie Blue G, 55Coomassie Brilliant Blue staining, 73, 497

maximal sensitivity, 769quick method, 768standard method, 768

Coomassie spot test for protein quantification,790

Copper chloride, 55Copper staining of gels, 75, 772Counterstaining cells (protocol), 729Counting myeloma or hybridoma cells

(protocol), 318–319, 319fCounting 32P or 125I from polyacrylamide gel

slices, 767Counting 35S, 14C, 3H from polyacrylamide gel

slices, 767Coupled antigens, pH of, 54Coupling

choosing strategy for, 53–54multiple sites, 54

Coupling antigens, 62–64to beads, 63bifunctional cross-linkers, 50thaptens, 50immune complexes as antigens, 63–64to protein carriers, 62protocols

coupling to red blood cells, 72modifying antigens by arsynyl coupling,

67–68modifying antigens by dinitrophenol

coupling, 66to red blood cells, 63with T-cell receptor–MHC-class II

protein-binding sites, 62–63Coverslips, growing adherent cells on (protocol),

686Cows

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgG binding affinity for Proteins A, G, and L,540t

CpG DNA, Magic Mouse adjuvant and, 115,146–147

Critical micelle concentration, 804Cross-linkers

bifunctional, 50t, 62removal of, 62selection for labeling antibody, 431–433,

432t, 433tamines/carbonyls, 431–432, 432t, 433tcarbohydrates (aldehydes), 432, 433tsulfhydryls, 432t, 433, 433t

Cross-linking peptides to KLH with maleimide(protocol), 85–86

Cross-reactivity, antibody, 6Cutting devices, safe handling of, 821Cyanogen bromide, 377Cyclophosphamide, in subtractive immuniza-

tion, 60Cy5-phycoerythrin, labeling antibodies with,

452–454Cysteine

maleimide linkage and, 54metabolic labeling of antigens with [35S]

methionine (protocol), 546–551Cytocentrifuge, attaching suspension cells to

slides using (protocol), 688Cytokines, 37, 38tCytometric Bead Assay (CBA), 232, 300Cytotoxicity

antibody-dependent cellular cytotoxicity(ADCC) by flow cytometry(protocol), 751–754, 752f–753f

complement-dependent cytotoxicity assay(protocol), 747–748, 748f

DDAB. See DiaminobenzidineDAB substrate solution for HRP (recipe), 523DAPI, properties of, 723tDavid’s life chart II, 788tDCC (dicyclohexylcarbodiimide), 431–432DEAE Affi-Gel Blue (Bio-Rad), 374–375DEAE chromatography

anion-exchange chromatography, 373–374high-pH DEAE chromatography (protocol),

391–392low-pH DEAE chromatography (protocol),

389–390Decoy immunization, 60

for mice, rats, and hamsters (protocol), 190Denaturation, modifying antigens by (protocol),

69Denaturing lysis (protocol), 588–590Dendritic cells, 2, 2f, 4, 33–34, 34f

transfected dendritic cell immunizations(protocol), 104–106

Desalting or buffer exchange usingsize-exclusion chromatography(protocol), 467–468

Detection of stained cellsalkaline phosphatase, 683t, 718–720b-galactosidase, 683t, 721double labeling, 682–683enzyme-labeled reagents, 681, 682t, 683, 683tfluorochrome-labeled reagents, 682, 682t,

722–724gold-labeled reagents, 682, 682t, 725–726horseradish peroxidase, 683t, 713–717iodine-labeled reagents, 682, 682t, 727–728overview, 681–683protocols

detecting alkaline phosphatase-labeledcells

using BCIP-NBT, 719–720using NABP-NF, 718

detecting b-galactosidase-labeled cellsusing X-gal (protocol), 721

detecting fluorochrome-labeled reagents,722–724

detecting gold-labeled reagents, 725–726detecting horseradish peroxidase-labeled

cellsusing aminoethylcarbazole, 717using chloronaphthol, 716using diaminobenzidine, 713–714,

714fusing diaminobenzidine and metal

salts, 715detecting iodine-labeled reagents, 727–728

Detergent lysisof animal tissues (protocol), 568–570differential of cellular fractions (protocol),

575–578of tissue culture cells (protocol), 565–567

Detergentsamphoteric, 805, 806tcritical micelle concentration, 804described, 804hydrophile-lipophile balance (HLB), 804ionic, 805, 806tmicelle molecular weight, 804nonionic, 805, 806tproperties of commonly used, 806tremoval of, 805

Developing solution for AP (recipe), 239, 241Developing solution for Strep-AP (recipe), 259Developmental Studies Hybridoma Bank, 309Dialysis for cleanup and purification of antibody

conjugates, 465–466Dialysis tubing preparation, 795Diaminobenzidine (DAB)

detecting horseradish peroxidase-labeledcells using (protocol), 713–714,714f

detecting horseradish peroxidase-labeledcells using diaminobenzidine andmetal salts (protocol), 715

substrate for horseradish peroxidase, 519, 522Diaminobenzidine (DAB) solution (recipe), 257Dibutyl phthalate

mounting cell or tissue samples in DPX(protocol), 730

Dicyclohexylcarbodiimide (DCC), 431–432Diethylene triamine pentaacetic acid (DTPA), 455Digital imaging, 733Digitonin, in differential detergent lysis of cell-

ular fractions protocol, 575–576Digitonin extraction buffer (recipe), 577Dimethyl pimelimidate (1�) (recipe), 596Dimethyl pimelimidate (DMP), crossing anti-

bodies to beads using, 591, 592f,593–596

Dimethyl sulfoxide (DMSO), 55, 309Dinitrophenol coupling, modifying antigens by

(protocol), 66Direct ELISA

advantages and disadvantages, 633fantibody capture assay, 211fcell-based ELISA, 661–665, 663f–664fdirect competitive ELISA, 666–669, 666fsandwich ELISA, 652f

Disposalgeneral cautions, 819–821of laboratory waste, 821

Distrenemounting cell or tissue samples in DPX

(protocol), 730

830 / Index

Disuccinimidyl glutarate (0.5 M) (recipe), 627Disuccinimidyl glutarate (DSG), 625–626Disuccinimidyl subpimelimidate (DSS), crossing

antibodies to beads using, 591,592f, 597–600

Disulfide bonding, 57Dithiothreitol (DTT), 474D-10 medium (recipe), 277DMEM (Dulbecco’s modified Eagle’s medium),

304, 305t–306tDMP (dimethyl pimelimidate), crossing anti-

bodies to beads using, 591, 592f,593–596

DMSO (dimethyl sulfoxide), 55, 309DMSO (dimethyl sulfoxide), for storage of

hybridomas, 309DNA. See also cDNA

CpG, 115, 146–147genetic immunization, 125–126rearrangements

heavy-chain genes, 15–16, 16fk gene and, 14–15, 15fl gene and, 15, 16f

DNA blotting, 470DNA loading buffer (6�) (recipe), 622DNASTAR software package, 51DNBS (sodium dinitrobenzene sulfonic acid), 66DNP. See DinitrophenolDOC (sodium deoxycholate), 535

in differential detergent lysis of cellularfractions protocol, 575–576

Dogsbinding characteristics of Protein A, Protein

G, and Protein L, 212tIgG binding affinity for Proteins A, G, and L,

540tDonkey

IgG binding affinity for Proteins A, G, and L,540t

DOTA (1,4,7,10-tetraazacyclododeane-tetraaceticacid), 455, 457

Dot blotsantibody capture assay, 213antibody capture on nitrocellulose

membrane (protocol), 242–243antigen capture assay: reverse dot blots

(protocol), 214apparatus, 213f

Dot plots, 736, 736fDounce homogenization, lysis using (protocol),

571–574DPX, mounting cell or tissue samples

(protocol), 730Drug selection, 218

unfused myeloma cells eliminated by, 205Drying gels, 773DSG (disuccinimidyl glutarate), 625–626DSS (disuccinimidyl subpimelimidate), crossing

antibodies to beads using, 591,592f, 597–600

DTPA (diethylene triamine pentaacetic acid), 455DTT (dithiothreitol), 474Dulbecco’s modified Eagle’s medium (DMEM),

304, 305t–306tDulbecco’s phosphate-buffered saline (pH 7.2,

1�) (recipe), 599Dyes, hazards of, 823

EEBC lysis buffer (recipe), 622ECF buffer 1� (recipe), 284

EDC (ethyl(dimethylaminopropyl)carbodiimide), 431–432

Effector domains, 362, 364EIA. See Enzyme immunoassayElectro cell fusion (protocol), 281–285, 282f

materials, 281method, 282–283

performing fusion, 283preparation of cells for fusion, 282–283purification of B cells from lymph nodes

by negative selection, 282recipes, 284–285setup, 282ftroubleshooting, 283–284

Electroelution of protein antigens frompolyacrylamide gel slices(protocol), 79–80

Electron microscopylabeling antibodies with colloidal gold

(protocol), 459–460Electrophorectic transfer to nitrocellulose

membranes (protocol), 81–82Electrophoresis, 755–775. See also Polyacryla-

mide gel electrophoresisalkylation of proteins before running on a

gel, 765autoradiography, 775Coomassie Brilliant Blue staining

maximal sensitivity, 769quick method, 768standard method, 768

copper staining of gels, 772counting 32P or 125I from polyacrylamide gel

slices, 767counting 35S, 14C, 3H from polyacrylamide

gel slices, 767drying gels, 773fixing gels, 773fluorography, 775partial proteolytic peptide maps, 760–761PPO fluorography, 766rehydrating dried polyacrylamide gels, 774SDS-polyacrylamide gel electrophoresis,

755–759silver staining of gels

ammoniacal silver staining, 770neutral silver staining, 771

two-dimensional isoelectric focusing/SDS-polyacrylamide gelelectrophoresis, 762–764

Electrophoretic transfer, 55semi-dry, 499–502, 500fwet, 503–506

Electroporation of cDNA into E. coli, 418–419ELISA. See Enzyme-linked immunosorbant assayELISpot assay, 651Elution buffer (recipe), 623Embedding cultured cells in matrigel (protocol),

701–702EMD Millipore, 409Engineering antibodies, 405–428

creation of recombinant antibodies usingdegenerate oligionucleotides(protocol), 407–409

discussion, 408–409materials, 407–408method, 408

creation of recombinant antibodies usingphage display (protocol),412–421

discussion, 420materials, 412–414

method, 414–420amplification of gene III, 415construction of cDNA library,

417–420, 419tconstruction of Gene III-kappa

c-region insert and ligation intopPDS, 415–416

construction of pLINK and pFABC,416–417

construction of pPDS vector, 414primer list, 419t

creation of recombinant antibodies using50-RACE (protocol), 410–411

materials, 410method, 410–411recipe, 411

modification of antibody function bymutagenesis (protocol), 422–423

materials, 422method, 423

rescue strategy for nonviable hybridomas(protocol), 424–427

materials, 424–425method, 425–427

Enzymatic detection of immunoblots, 477Enzyme immunoassay (EIA), 629. See also

Enzyme-linked immunosorbantassay

Enzyme-linked immunosorbant assay (ELISA),629–673. See also specific ELISAtypes

antibody detection and quantification, 632antigen detection and quantification,

630–631bead-based, 642–643blocking buffers, 635–636

BLOTTO, 635bovine serum albumin (BSA)-based, 635casein-based, 636gelatin, 636protein-free, 636serum, 636

checkerboard titration of antigen andantibody by serial dilution, 654t

choosing a protocol, 631tdeciding where to start, 630ELISpot assay, 651epitope binding competition assays, 355, 356fhost cell protein (HCP), 632, 655–656indirect versus direct detection, 632, 633fisotyping and, 361, 366–370isotyping template for, 295f, 298fto measure antibody affinity, 358–359microtiter plate selection, 632–635, 634f, 634tmultiplexing flow cytometry and, 737overview, 629–643parameters, 639–640, 639fprotocols, 644–673

direct and indirect cell-based ELISA,661–665, 663f–664f

direct competitive ELISA, 666–669, 666fimmunometric antibody sandwich

ELISA, 651–657, 652f, 654t,655f–656f

immunometric double-antibody sand-wich ELISA, 658–660, 658f

immunometric indirect ELISA, 644–650,644f–645f, 647t–649t

indirect competitive ELISA, 670–673,671f, 673f

reporter-labeled secondary antibodies,636–637, 637t

Index / 831

Enzyme-linked immunosorbant assay (ELISA)(Continued)

screening forphospho-specificantibodies,214substrates, 637–639

chemifluorescent, 638–639chemiluminescent, 638chromogenic (colorimetric), 637–638

troubleshooting guide, 647t–648ttypes, 642–643validation parameters, 640–642, 640t

accuracy, 641limit of detection (LoD), 641linearity, 641precision, 640quantification limit (LoQ), 641range, 642sensitivity, 641specificity, 642

wash buffers, 636Enzyme-linked immunosorbant spot (ELISpot)

assay, 651Epinephrine, for anaphylactic shock, 122Epitope

amino acid mobility an, 52antibody specificity and, 353–354binding competition assays, 355, 356fb-turns in, 52choosing between recombinant proteins and

peptides for immunogenproduction, 57

conformational, 354–355described, 6, 354–357discontinuous, 57functional, 355heat-induced epitope retrieval (protocol),

698–699linear, 354–355mapping, 355–357processed antigen fragments, 35–36size of, 22, 53structural, 355structure of, 22–23tags in immunoprecipitation, 539

Epitope retrieval, heat-induced (protocol),698–699

Epstein-Barr virus (EBV), 234Equilibrium dialysis to obtain affinity constants

for antibodies, 358Escherichia coli

electroporation of cDNA into, 418–419for recombinant protein expression, 58, 58t

Ethanolamine, 593–594Ethanolamine (1�) (recipe), 596Ethyl(dimethylaminopropyl) carbodiimide

(EDC), 431–432Europium, labeling antibodies using, 457–458Euthanasia

killing mice, rats, and hamsters using carbondioxide asphyxiation (protocol),194–195

ExactSTART, 410ExPASy, 51Exsanguination, 129Extracellular proteins, designing peptides against

highly homologous, 53

FFab, 14

fragments, 10, 11fpurification of Fab and F(ab0)2

fragments, 376

FACSintracellular staining by, 249–250isotyping and, 361surface staining by flow cytometry/FACS,

246–248FACS buffer (recipe), 745FastPrep Cell Disruptor, 580Fast protein liquid chromatography (FPLC),

373–374FBS. See Fetal bovine serumFc-binding proteins, human isotype classes and,

363tFc domains, immunoglobulin isotypes and,

363–364, 363fFc fragments, 10–11, 11fFcRn (neonatal Fc receptor), 236Feeder cells

fibroblast feeder cell culture preparation(protocol), 271

splenocyte feeder cell culture preparation(protocol), 269–270

Feeder plate preparation, 218myeloma cell, 267–268peritoneal macrophage, 266

Fentanyl/droperidol for anesthesia, 112Fentanyl/fluanisone for anesthesia, 112Fentanyl for anesthesia, 112, 140Fetal bovine serum (FBS)

addition to cell culture media, 304screening for good batches of, 264–265

FiberCell Systems, 315, 315fFibroblast feeder cell culture preparation

(protocol), 27150-RACE, creation of recombinant antibodies

using (protocol), 410–411materials, 410method, 410–411recipe, 411

Fixation, 680fixing attached cells (protocols)

in organic solvents, 703–704in paraformaldehyde or glutaraldehyde,

705–706fixing suspension cells with paraformaldehyde

(protocol), 707of gels, 773overview of, 680unmasking hidden epitopes with proteases,

680FLAG

epitope tag, 539, 607tandem immunoaffinity purification using

anti-FLAG and anti-HAantibodies (protocol), 607–612

Flow cytometry, 735–754data plots, 736–737, 736fdetermining the class and subclass of a

monoclonal antibody using(protocol), 300–301

equipment, 735fluorochrome excitation and emission

spectra, 736, 736fintracellular staining by, 249–250multiplexing, 737, 740overview of, 735–737plate-based, 738, 739tprotocols, 742–754

antibody-dependent cellular cytotoxicity(ADCC), 751–754, 752f–753f

apoptosis assessment, 749–750, 750fcomplement-dependent cytotoxicity

assay, 747–748, 748f

simple multiplexed antibody-bindingassay, 742–745, 744f, 744t

screening assay design and optimization,738, 740

surface staining by flow cytometry/FACS,246–248

Fluoresceindetecting fluorochrome-labeled reagents in

stained cells (protocol), 722–724labeling antibodies using NHS-fluorescein

(protocol), 444–445properties of, 723t

Fluorescence. See also Flow cytometry; specificprotocols

chemifluorescent substrates, ELISA,638–639

immunofluorescenceantibody capture on permeabilized

whole cells, 253–254surface staining by, 251–252

quenching, 733Fluorescent proteins

protein–antibody conjugate formation,448–449, 452–454

Fluorescent resonance energy (FRET) dyes,448–449, 452–453

Fluorochromes. See also Fluorophoreschoosing correct, 723–724detecting fluorochrome-labeled reagents in

stained cells, 722–724for detection of antigen on immunoblots,

519–520, 525excitation and emission spectra of, 723t, 736,

736ffluorochrome-labeled secondary reagents for

immunoblotting, 478fluorochrome-labeled strepavidin, 516–517properties of, 723treprobing membranes after immunoblotting,

526Fluorography, 766, 775Fluorophores. See also Fluorochromes

labeling antibodies with, 442–447overview, 442–443, 442tusing a maleimido dye (protocol),

446–447using NHS-fluorescein (protocol),

444–445list of fluorophores, 442t

Footpad immunization, 123of mice, rats, and hamsters (protocol),

168–169, 169fFootprinting, antibody, 356Formaldehyde, for cross-linking proteins to

DNA, 614, 617–618, 625–626Formaldehyde solution (11%) (recipe), 623FPLC (fast protein liquid chromatography),

373–374Fragmenting a wet gel slice (protocol), 77Freezing

cell pellets for large-scaleimmunoprecipitation (protocol),563–564

cells for liquid nitrogen storage, 321–322for storage of hybridomas, 309

FRET (fluorescent resonance energy) dyes,448–449, 452–453

Freund’s adjuvant, 113, 114–115advantages and disadvantages of, 114tascites induction using Freund’s adjuvant

(protocol), 182Complete, 113, 114, 142

832 / Index

Incomplete, 114preparation protocol, 142–143, 143f

Frozen tissue sections, preparing (protocol),693–695

Functional assays, 215–216Functional epitope mapping methods, 355–357Functional screens, 210f, 214–215

for blocking/neutralizing or activatingantibodies, 215

for internalizing antibodies, 215, 216ffor phospho-specific antibodies, 214–215

Fungi contamination of cell cultures, 230f,309–310

overview, 309–310ridding cell lines of by antibiotics (protocol),

325–326ridding cell lines of by passage through mice

(protocol), 328–329ridding cell lines of with peritoneal

macrophages (protocol), 327Fusion protein preparation protocols

GST-fusion proteins, 87–88His-fusion proteins, 89–91MBP-fusion proteins, 92–94mFc- and hFc-fusion protein preparation

from mammalian cells, 97–99Fusions

back-fusing, 229overview of, 220–222

polyethylene glycol, 222Sendai virus, 221

protocolselectro cell, 281–285, 282fpolyethylene glycol, 274–278by sendai virus, 279–280

Fusion selection (HAT) medium (recipe), 268

GGas containers, safe handling of, 820Gateway expression vectors, 817fGelatin, for ELISA blocking, 636Gel fragments, processing, 55Gels

drying, 773fixing, 773rehydrating dried, 774transfer of proteins from gels to membranes,

497–508overview, 497semi-dry electrophoretic transfer

(protocol), 499–502, 500fstaining the blot for total protein with

Ponceau S (protocol), 507–508wet electrophoretic transfer (protocol),

503–506Gel slice(s)

counting 32P or 125I from polyacrylamide gelslices, 767

counting 35S, 14C, 3H from polyacrylamidegel slices, 767

electroelution of protein antigens frompolyacrylamide (protocol), 79–80

fragmenting (protocol), 77lyophilization (protocol), 8

Gelvatol, mounting cell or tissue samples in(protocol), 731

Gene IIIamplification of, 415construction of Gene III-kappa c-region

insert and ligation into pPDS,415–416

Gene knockout, immune response weakened by,53

Genetic code, 781tmitochondrial, 782t

Genetic immunization, 125–126GenomOne Kits, 279Gentamicin, addition to culture media, 304, 306tGERBU adjuvant, 116Germinal centers, 39GFP (green fluorescent protein), 448Glass beads, lysing yeast cells with (protocol),

579–581using lysis buffer 2 without detergents

(protocol), 582–584Glutaraldehyde, 432

fixing attached cells in (protocol), 705–706Glutathione-S-transferase (GST), 55, 56t

as affinity tag, 539GST-fusion protein preparation from

bacteria (protocol), 87–88Glycerol

for antibody storage, 383for hybridoma storage, 309

Glycine, 597, 599, 608, 609Glycoproteins, antigen receptors as, 5Goats

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgG binding affinity for Proteins A, G, and L,540t

Goldcolloidal, 455, 459–460detecting gold-labeled reagents, 725–726

Green fluorescent protein (GFP), 448Growing adherent cells (protocols)

on coverslips or multiwell slides, 686on tissue culture dishes, 687

Growing hybridomas. See Hybridomas, growingGST. See Glutathione-S-transferaseGST elution buffer, pH 8.0 (recipe), 88GST-fusion protein preparation from bacteria

(protocol), 87–88GST lysis buffer (recipe), 88Guanidine-chloride stripping buffer (recipe), 529Guinea pigs

antigen dose for, 116binding characteristics of Protein A, Protein

G, and Protein L, 212tbleed sample amount from, 110IgG binding affinity for Proteins A, G, and

L, 540t

HHA (hemagglutinin)

epitope tag, 539, 607tandem immunoaffinity purification using

anti-FLAG and anti-HAantibodies (protocol), 607–612

Haloacetyl cross-linkers, 433, 433tHam’s F-12 medium, 305t–306tHamsters

administering anesthesia to (protocol),136–138, 137f

antigen dose for, 116, 118tbleed sample amount from, 110cDNA immunization (protocol), 192–193, 193fdecoy immunization for (protocol), 190footpad or hock immunization (protocol),

168–169, 169fIgG binding affinity for Proteins A, G, and L,

540t

injection routes for, 120tkilling using carbon dioxide asphyxiation

(protocol), 194–195lymph node harvesting from (protocol),

198–199, 199frepetitive immunization at multiple sites

(RIMMS) (protocol), 188spleen harvesting from (protocol), 196–197,

197fstandard immunization (protocol),

184–185subtractive immunization (protocol), 189test bleeds in, 128–129

Haptens, 36, 50cross-linking peptides to KLH with

maleimide (protocol), 85–86Harvesting tissue protocols, 194–199

killing mice, rats, and hamsters usingcarbon dioxide asphyxiation,194–195

lymph node harvesting from mice, rats, andhamsters, 198–199, 199f

spleen harvesting from mice, rats, andhamsters, 196–197, 197f

HAT fusion medium (recipe), 278, 284HAT (hypoxanthine/aminopterin/thymidine)

selection, 217, 218, 224, 267HAT selection medium (recipe), 351Hazardous chemicals, properties of common,

823–824HCP (host cell protein) ELISA, 632Heat-induced epitope retrieval (protocol),

698–699Heavy-chain gene, DNA rearrangements and,

15–16, 16fHeavy metal chelators, 455HEK-Blue Detection medium, 338–339, 339fHelper T cells, 35, 36Hemagglutinating virus of Japan (HVJ), 221,

279–280. See also Sendai virusHemagglutinin (HA)

epitope tag, 539, 607tandem immunoaffinity purification using

anti-FLAG and anti-HAantibodies (protocol), 607–612

Hemocytometer, 318–319, 319fHeterokaryon, 205High-pressure liquid chromatography (HPLC),

373–374High-throughput functional assays

antibody-dependent cellular cytotoxicity(ADCC), 751–754, 752f–753f

apoptosis assessment, 749–750, 750fcomplement-dependent cytotoxicity assay,

747–748, 748fHigh-throughput western blot assay for

hybridoma screening(protocol), 244–245

Hinge region, antibody, 10, 11fHis6 epitope tag, 539His-fusion binding buffer, pH 7.4 (recipe), 90His-fusion elution buffer, pH 7.4 (recipe), 90His-fusion lysis buffer, pH 7.4 (recipe), 91His-fusion protein preparation from bacteria

(protocol), 89–91Histograms, 736, 736fHLB (hydrophile-lipophile balance), 804HMT selection medium (recipe), 351Hock immunization

injection into hock, 123–124of mice, rats, and hamsters (protocol),

168–169, 169f

Index / 833

Hoechst dye 33258 stainingproperties of fluorochrome, 723ttesting for mycoplasma contamination by,

332–333, 333fHollow-fiber reactors, 314–316, 315fHomemade chemiluminescent substrate

solution for HRP: solution A(recipe), 523

Homemade chemiluminescent substratesolution for HRP: solution B(recipe), 524

Homologous protein, designing peptides against,53

Horseradish peroxidase (HRP)in antibody capture assays, 210–211, 232,

242–245antibody conjugation to, 448, 450–451in antigen capture assays, 261–262in antigen detection on immunoblots, 519,

521–524detecting horseradish peroxidase–labeled

cellsusing aminoethylcarbazole, 717using chloronaphthol, 716using diaminobenzidine, 713–714, 714fusing diaminobenzidine and metal salts,

715Horses

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgG binding affinity for Proteins A, G, and L,540t

Host cell protein (HCP) ELISA, 632,655–656

HPLC (high-pressure liquid chromatography),373–374

HPRT. See Hypoxanthine-guanine phosphori-bosyl transfer (HPRT) gene

HRP. See Horseradish peroxidaseHT (hypoxanthine/thymidine) selection, 218,

224, 226Human hybridomas, 234–235Human isotype classes and associated

Fc-binding proteins, 363tHumanization technology, 235Humans

binding characteristics of Protein A, ProteinG, and Protein L, 212t

IgG binding affinity for Proteins A, G, and L,540t

isotype classes and associated Fc-bindingproteins, 363t

Hunter’s TiterMax adjuvant, 114t, 115, 145HVJ (hemagglutinating virus of Japan), 221,

279–280Hybridoma(s). See also Monoclonal antibodies

cell repositories, 309description of, 203feeding, 224growing (see Hybridomas, growing)history of development, 202, 203human, 234–235interspecies, 205, 234monoclonal antibody production by, 48production (see Hybridoma production)rescue strategy for nonviable hybridomas

(protocol), 424–427materials, 424–425method, 425–427

selecting best from fusion, 44Hybridoma Cloning Factor, 217, 224, 226–227,

269–270, 286–290

Hybridoma cloning supplements, 217Hybridoma production

chromosome loss, 205class-switching variants, selecting, 233–234cloning of hybridoma cells, 228contamination, dealing with, 229–231

of a cloned line, 230–231common forms of contamination, 230fin fusion wells, 229–230

expanding and freezing positive clones,225–229, 227f

single-cell cloning, 228unstable lines, 229

feeding hybridomas, 224fusions, 220–222

elimination of unfused cells by drugselection, 205, 206f

overview of, 220–222polyethylene glycol as fusing agent,

205, 222Sendai virus use, 221

future trends, 235–236antibody optimization, 235humanization technology, 235next generation mice, 236single-cell PCR technology, 235synthetic antibodies, 235–236

human hybridomas, 234–235injection routes for, 120interspecies hybridomas, 234isotyping and subclassing of monoclonal

antibodies, 231–234myeloma from BALB/c mice, 204–205overview of, 216–217plating strategies, 222–224

bulk plating, 223multiple cells per well, 223, 223fsingle cell per well, 222–223, 222fsoft agar use, 224, 224f

preparation for fusions, 217–220drug selections, 218feeder layer plate preparation, 218final boost, 218–220hybridoma cloning supplements, 217low-density growth supplements, 217preparing myeloma cells for fusions, 219,

219fpreparing partner cells for fusions,

218–219preparing splenocytes for fusions, 219,

219f–221frecombinant murine IL-6, 217

progression of hybridoma growth postfusion,226f

screening method, 207–216, 225antibody capture assays, 209–213,

210f–211f, 212t, 213fantibody capture on nitrocellulose

membrane: high-throughputwestern blot assay for hybridomascreening (protocol), 244–245

antigen capture assays, 210f, 213–214characteristics of a good procedure,

207development of, 207–216examples of, 225functional assays, 215–216functional screens, 210f, 214–215

for blocking/neutralizing oractivating antibodies, 215

for internalizing antibodies, 215,216f

for phospho-specific antibodies,214–215

pooling strategies, 225supernatant collection strategies for

screening, 224–225timing of screening, 207

stages of, 207, 208f–209funstable, 205, 229

Hybridomas, growing, 303–352cell culture, 304–309

antibiotics for, 304, 306tlong-term storage for, 309media, 304–307, 305t–306tsending and receiving hybridomas and

myelomas, 309suppliers of media, 307ttechnique for, 307–308, 308f

contamination of cell cultures, 309–313by bacteria, fungi, or yeast, 309–310by mycoplasma, 310–313, 311t–312t

drug selection, 316–317checking cell lines for HGPRT

deficiency, 316, 350–351selecting HGPRT mutants with

8-azaguanine, 316–317, 349monoclonal antibody production and

storage, 313–316collecting tissue culture supernatants,

313–314comparison of methods, 313thollow-fiber reactors, 314–316, 315fIntegra CELLine 1000 flask, 314, 314fpreparing tissue culture supernatants

with higher concentrations ofantibody, 314–316, 314f–315f

roller bottles and large T-flasks, 314overview, 303–317protocols, 318–352

ascites induction and collection,344–345

checking cell lines for HPRT deficiency,350–352

collecting tissue culture supernatants,346

counting myeloma or hybridoma cells,318–319, 319f

freezing cells for liquid nitrogen storage,321–322

recovering cells from liquid nitrogenstorage, 323–324

ridding cell lines of contaminatingmicroorganisms by antibiotics,325–326

ridding cell lines of contaminatingmicroorganisms by passagethrough mice, 328–329

ridding cell lines of contaminatingmicroorganisms with peritonealmacrophages, 327

ridding cells of mycoplasmacontamination by passagethrough mice, 342–343

ridding cells of mycoplasma contamina-tion using antibiotics and single-cell cloning, 340–341

selecting myeloma cells for HGPRTmutants with 8-azaguanine, 349

storing tissue culture supernatants andascites, 347–348

testing for mycoplasma contaminationby growth on microbial media,330–331, 331f

834 / Index

testing for mycoplasma contaminationby Hoechst dye 33258 staining,332–333, 333f

testing for mycoplasma contaminationusing PCR, 334–337, 335f

testing for mycoplasma contaminationusing reporter cells, 338–339,339f

viability checks, 320Hydrazide, 432, 433t

biotinylating antibodies usinghydrazide-LC-biotin (protocol),440–441

Hydrazine, 377Hydrophile-lipophile balance (HLB), 804Hydrophilic amino acid exposure on protein

surface, 52Hydrophilic peptides, advantages of, 52Hydroxyapatite chromatography, 374–375Hydroxylamine buffer (recipe), 447, 451Hydroxysuccinimide. See N-hydroxysuccinimide

(NHS)HyperCyt, 738, 739tHyperimmunization, 126–127Hypervariable regions, 14Hypoxanthine/aminopterin/thymidine (HAT)

selection, 217, 218, 224, 267Hypoxanthine-guanine phosphoribosyl transfer

(HPRT) gene, 205, 206fchecking cell lines for HPRT deficiency, 316,

350–352selecting myeloma cells for HGPRT

mutants with 8-azaguanine,316–317, 349

Hypoxanthine/thymidine (HT) selection, 218,224, 226

IIDA (iminodiacetic acid), 375IgA. See Immunoglobulin AIgD. See Immunoglobulin DIgE. See Immunoglobulin EIgM. See Immunoglobulin MIgY. See Immunoglobulin YIL-6, 34IMAC (immobilized metal affinity

chromatography), 89–91Imaging, digital, 733IMDM medium, 305t–306tIMG-AbS platform mice, 236Iminodiacetic acid (IDA), 3752-Iminothiolane (Traut’s Reagent), 433Immobilized metal affinity chromatography

(IMAC), 89–91, 375ImmunEasy adjuvant, 114tImmune complex. See also Antibody–antigen

complexas antigens, 63–64conformational changes in, 23preparation for injection (protocol),

70–71stabilization by noncovalent bonds, 23

Immune exhaustion, 6Immune response

failure to induce, 46–47role in combating microbial invasion, 1–2weakened by gene knockout, 53

Immune systemantibody production by the, 1–7overview of, 1–7

Immune tolerance, 6, 37–39

Immunization. See also Immunization protocols;Immunizing animals

cDNA, 61, 192–193, 193fdecoy, 60, 190live cell preparation (protocol), 100subtractive, 60, 189transfected dendritic cell immunizations

(protocol), 104–106Immunization protocols

devising of, 131–132protocols, 182–193

adoptive transfer immunization ofmice, 191

ascites induction in BALB/c mice usingmyeloma cells, 183

ascites induction using Freund’sadjuvant, 182

cDNA immunization mice, rats, andhamsters, 192–193, 193f

decoy immunization for mice, rats, andhamsters, 190

repetitive immunization at multiple sites(RIMMS) of mice, rats, andhamsters, 188

spleen harvesting from mice, rats, andhamsters, 196–197, 197f

standard immunization of mice, rats, andhamsters, 184–185

standard immunization of rabbits,186–187

subtractive immunization for mice, rats,and hamsters, 189

selection of, 130–131, 131tImmunizing animals, 107–199

adjuvants, 113–116advantages and disadvantages of specific,

114taluminum hydroxide, 116Freund’s, 114–115, 142–143, 143fGERBU, 116Hunter’s TiterMax, 115, 145Magic Mouse, 115, 146–147overview of, 113–114Pam3Cys-Ser-(Lys)4, 115–116protocols, 142–149Ribi, 115, 144

anesthesia, 112, 113tprotocols, 136–141

animal choice for, 109–110ascites fluid induction in mice, 129dose of antigen, 116–118

for mice, 117tfor rabbits, 117tfor rats and hamsters, 118t

exsanguination, 129form of immunogen, 118–119

carbohydrates, 119insoluble, 118–119live cells, 119nucleic acids, 119recombinant proteins, 119soluble, 118synthetic peptides, 119

immunization protocoldevising of, 131–132protocols, 182–193selection of, 130–131, 131t

legalandmoralconsiderationsofuse,108–109number of animals needed, 112overview, 108, 130polyclonal versus monoclonal antibodies,

109–110, 109t, 110t

protocols, 136–199anesthesia and adjuvants, 136–149

administering anesthesia to mice,rats, and hamsters, 136–138, 137f

administering anesthesia to rabbits,139–141

aluminum hydroxide (alum)adjuvant preparation, 148–149

Freund’s adjuvant preparation,142–143, 143f

Hunter’s TiterMax adjuvant use, 145Magic Mouse adjuvant use, 146–147Ribi adjuvant use, 144

harvesting tissue, 194–199killing mice, rats, and hamsters using

carbon dioxide asphyxiation,194–195

lymph node harvesting from mice,rats, and hamsters, 198–199, 199f

spleen harvesting from mice, rats,and hamsters, 196–197, 197f

immunization protocols, 182–193adoptive transfer immunization of

mice, 191ascites induction in BALB/c mice

using myeloma cells, 183ascites induction using Freund’s

adjuvant, 182cDNA immunization mice, rats, and

hamsters, 192–193, 193fdecoy immunization for mice, rats,

and hamsters, 190repetitive immunization at multiple

sites (RIMMS) of mice, rats, andhamsters, 188

standard immunization of mice, rats,and hamsters, 184–185

standard immunization of rabbits,186–187

subtractive immunization for mice,rats, and hamsters, 189

routes of antigen injection, 150–169footpad or hock immunization of

mice, rats, and hamsters,168–169, 169f

immunizing mice and rats withnitrocellulose-bound antigen,154–156, 155f

intradermal injection in rabbits,159–160, 160f

intramuscular injection in rabbits,157–158, 158f

intraperitoneal injection with adju-vant in mice and rats, 165, 165f

intraperitoneal injection withoutadjuvant in mice and rats,166–167, 166f–167f

intravenous injection in mice,163–164, 164f

intravenous injection in rabbits,161–162, 162f

subcutaneous injection in rabbits,150–151

subcutaneous injection with adjuvantinto mice and rats, 152

subcutaneous injection withoutadjuvant into mice and rats, 153

serum sampling and preparation,170–181

sampling mouse and rat serum fromretro-orbital sinus, 175–176,176f

Index / 835

Immunizing animals, (Continued)sampling mouse and rat serum from

saphenous vein, 179–180, 180fsampling mouse and rat serum

from submandibular vein,177–178, 177f

sampling mouse serum from tailvein, 172–173, 173f

sampling rabbit serum from marginalear vein, 170–171, 170f

sampling rat serum from tailvein, 174

serum preparation, 181routes of injection, 120–127

affinity maturation and, 127anaphylactic shock and, 122boosts, 124clonal dominance and, 127decision on boost versus fusion, 126genetic (naked DNA) immunization, 125hyperimmunization and, 126–127intradermal, 121–122, 159–160, 160fintramuscular, 121, 157–158, 158fintraperitoneal, 122–123, 165–167,

165f–167fintravenous, 122, 161–164, 162f, 164finto lymphoid organs, 123–124for mice, rats, and hamsters, 120toverview, 120protocols, 150–169for rabbits, 121trepetitive immunization at multiple sites

(RIMMS), 124, 125f, 125tsubcutaneous, 120–121, 150–153

sampling serum, 127–129, 128t, 170–181sampling mouse and rat serum from

retro-orbital sinus, 175–176,176f

sampling mouse and rat serum fromsaphenous vein, 179–180, 180f

sampling mouse and rat serum fromsubmandibular vein, 177–178,177f

sampling mouse serum from tail vein,172–173, 173f

sampling rabbit serum from marginalear vein, 170–171, 170f

sampling rat serum from tail vein, 174serum preparation, 129, 181species/strain choice, 110–112, 110t, 111ttroubleshooting, 132–135

project 1, 132–133project 2, 133project 3, 133–134project 4, 134project 5, 134–135

Immunoadsorbants, 539–541, 540tImmunoaffinity purification using anti-FLAG

and anti-HA antibodies(protocol), 607–612

discussion, 611materials, 607–608method: anti-FLAG purification, 607–608method: anti-HA purification, 609–610recipes, 612troubleshooting, 610t–611t

Immunoassays, 629–673antibody detection and quantification, 632antigen detection and quantification,

630–631bead-based ELISA, 642–643blocking buffers, 635–636

BLOTTO, 635bovine serum albumin (BSA)-based, 635casein-based, 636gelatin, 636protein-free, 636serum, 636

choosing a protocol, 631tdeciding where to start, 630ELISA parameters, 639–640, 639fELISA validation parameters, 640–642, 640t

accuracy, 641limit of detection, 641linearity, 641precision, 640quantification limit, 641range, 642sensitivity, 641specificity, 642

indirect versus direct detection, 632, 633fmicrotiter plate selection, 632–635, 634f, 634toverview, 629–643protocols, 644–673

direct and indirect cell-based ELISA,661–665, 663f–664f

direct competitive ELISA, 666–669, 666fimmunometric antibody sandwich

ELISA, 651–657, 652f, 654t,655f–656f

immunometric double-antibody sand-wich ELISA, 658–660, 658f

immunometric indirect ELISA, 644–650,644f–645f, 647t–649t

indirect competitive ELISA, 670–673,671f, 673f

reporter-labeled secondary antibodies,636–637, 637t

substrates, 637–639chemifluorescent, 638–639chemiluminescent, 638chromogenic (colorimetric), 637–638

types, 642–643wash buffers, 636

Immunoblotting, 469–530antibody choice, 471–473, 472t

monoclonal antibodies, 472t, 473polyclonal antibodies, 472–473, 472t

detection methods, 473, 474tchromogenic, 477enzymatic, 478fluorochrome-labeled secondary

reagents, 478selection of, 477–478, 477fsignal amplification scale, 478

experimental strategies, 473–478adjusting the primary and secondary

antibody concentration, 476–477blocking nonspecific binding sites on the

membrane, 475–476, 476tdetection method selection, 477–478immunoblotting of the immunoprecipi-

tated proteins, 475, 476foverview, 473–474sample preparation, 474–475

optimizing primaryantibodydilutionfor, 476foverview, 470–478protocols, 479–530

blocking and incubation with antibodies,509–518

immunoblots prepared with immu-noprecipitated protein antigens(immunoprecipitation/westernblotting), 515–518

immunoblots prepared with whole-cell lysates and purified proteins(straight western blotting),510–514

overview, 509detection, 519–525

enzyme-based, 519, 521–524with fluorochromes, 519–520, 525

reprobing membrane after immunoblot-ting, 526–530

overview, 526stripping the blot for reprobing,

527–530resolving proteins by gel electrophoresis,

490–496materials, 491–492method, 492–494overview, 490recipes, 495–496troubleshooting, 494–495

sample preparation, 479–489preparing immunoprecipitations for

immunoblotting, 487–489preparing protein solution for

immunoblotting, 484–486preparing whole-cell lysates for

immunoblotting, 480–483transfer of proteins from gels to

membranes, 497–508overview, 497semi-dry electrophoretic transfer,

499–502, 500fstaining the blot for total protein

with Ponceau S, 507–508wet electrophoretic transfer, 503–506

schematic of classical procedure, 470fsteps in, 471strength of antibody binding, 25t

ImmunoCruz series, 509Immunodepletion of serum, 379–381,

379f–380f, 382fImmunodiffusion. See Ouchterlony double-

diffusion assaysImmunofluorescence

antibody capture on permeabilized wholecells, 253–254

surface staining by, 251–252Immunogen(s). See also Antigen(s)

accumulation in lymphoid organ, 4chemical features of, 46, 46tcoupling antigens, 50t, 62–64defined, 2form for immunizing animals, 118–119

carbohydrates, 117t, 118t, 119dose of antigen, 116–118, 117t, 118tinsoluble proteins, 117t, 118–119, 118tlive cells, 117t, 118t, 119nucleic acids, 117t, 118t, 119particulate proteins, 117t, 118trecombinant proteins, 119soluble proteins, 117t, 118, 118tsynthetic peptides, 119

properties of good, 45–46, 46tsize limit, 45, 53sources of, 49–61, 49t

cDNA, 49t, 61cell and tissue lysates, 49t, 59–60haptens, 50purified native proteins, 49t, 54–55recombinant proteins, 49t, 55–59synthetic peptides, 49t, 50–54

ImmunoGenes, 236

836 / Index

Immunogenicity, 44–49antigen valency affect on, 119defined, 2, 44failure of animal to respond, 46–47polyclonal versus monoclonal antibodies,

47–48, 47tproperties of good immunogens, 45–46, 46tof pure antigens (purified native proteins), 54selecting the antigen and, 48, 49tstrengthening, 61–64synthetic class II–T-cell receptor sites, 47t

Immunoglobulin(s). See also Antibodyclass-switching, 18f, 19, 39, 233–234defined, 2structures of, 232f

Immunoglobulin A (IgA)characteristics of, 13tFc-binding proteins, 363tfunction, 362structure, 12, 232f, 362subclasses, 10

Immunoglobulin D (IgD)characteristics of, 13tfunction, 362structure, 232f

Immunoglobulin E (IgE)characteristics of, 13tFc-binding proteins, 363tfunction, 362, 364structure, 232f

Immunoglobulin G (IgG)affinity for antigen, 39anion-exchange chromatography, 373–374binding affinity for proteins A, G, and L,

539–540, 540tcharacteristics of, 13tchicken, 376class-switching, 233–234cross-linking antibodies to beads, 591–592Fc-binding proteins, 363tfunctions of, 362, 364high-pH DEAE chromatography (protocol),

391–392in hyperimmune sera, 126–127isolation of a total IgG fraction, 372–373

ammonium sulfate precipitation(protocol), 372–373, 385–386

caprylic acid IgG isolation (protocol),373, 387–388

isotyping, 361–362low-pH DEAE chromatography (protocol),

389–390Protein A purification, 393–394Protein G purification, 395–396purification of Fab and F(ab0)2 fragments, 376secondary response and, 33structure, 10–11, 12–13, 232fsubclasses, 10

Immunoglobulin M (IgM)affinity for antigen, 39characteristics of, 13tclass-switching, 233–234Fc-binding proteins, 363tfunction, 362, 364primary response and, 32purification, 376–377structure, 11–13, 232f

Immunoglobulin Y (IgY)described, 375–376isolation of IgY from chicken eggs:

polyethylene glycol method(protocol), 399–400

isolation of IgY from chicken eggs: sodiumsulfate method (protocol),397–398

Immunohistochemistryantibody capture on tissue sections

(protocol), 255–257Immunological memory, 4, 6Immunometric antibody sandwich ELISA,

651–657, 652f, 654t, 655f–656fImmunometric double-antibody sandwich

ELISA, 658–660, 658fImmunometric indirect ELISA, 644–650,

644f–645f, 647t–649tImmunoprecipitated proteins

immunoblotting of, 475blocking and incubation with antibodies:

(protocol), 515–518preparing for immunoblotting (protocol),

487–489visualizing, 541–542

Immunoprecipitation, 531–627antibodies

monoclonal, 538polyclonal, 537–538

antigen–antibody complex formation, 538antigen capture assay, 214antigen capture in solution (protocol), 263background problems

nonspecific background, 543, 604tspecific background, 543, 604t

cell lysisdenaturing lysis (protocol), 588–590detergent lysis of animal tissues

(protocol), 568–570detergent lysis of tissue culture cells

(protocol), 565–567differential detergent lysis of cellular

fractions (protocol), 575–578freezing cell pellets for large-scale

immunoprecipitation (protocol),563–564

lysing yeast cells using a coffee grinder(protocol), 585–587

lysing yeast cells with glass beads(protocol), 579–581

using lysis buffer 2 without deter-gents (protocol), 582–584

lysis using dounce homogenization(protocol), 571–574

overview, 534–537lysis buffers, 534–535lysis of tissue, 536–537lysis of tissue culture cells, 535–536lysis of yeast, 537protease inhibitor use, 535, 536t

protocols, 565–590cell preparation, 534chromatin (ChIP)

dual cross-linking for (protocol),625–627

overview, 614protocol, 617–624

blocking and antibody coating ofmagnetic beads, 620

chromatin preparation, 619chromatin quality and quantity

determination and inputchromatin preparation, 619

cross-linking of DNA and protein, 618discussion, 621–622DNA purification, amplification, and

sequencing, 621

elution, 620materials, 617–618method, 618–621recipes, 622–624reverse cross-linking, 621setting up the ChIP, 620troubleshooting, 621twashing, 620

coimmunoprecipitation, 532controls, 543–544cross-linking antibodies to beads

overview, 541, 591–592, 592fusing dimethyl pimelimidate (DMP),

591, 592f, 593–596using disuccinimidyl subpimelimidate

(DSS), 591, 592f, 597–600epitope tags, 539experimental strategies, 533fimmunoadsorbants, 539–541, 540tmagnetic beads, 793optimization, 542–543overview, 532–534, 533fphases of technique, 532, 533fprotocol for, 602–606

discussion, 605materials, 602methods, 602–603recipes, 606troubleshooting, 604t–605t

radiolabeling of protein antigensmetabolic labeling of antigens with [32P]

orthophosphate (protocol),558–562

discussion, 561materials, 558–559method, 559–561formonolayer-culturedcells,559–560recipes, 561–562for suspension-cultured cells,

560–561metabolic labeling of antigens with [35S]

methionine (protocol), 546–551discussion, 549–550materials, 546–547method, 547–549recipes, 550–551suggested labeling amounts, 547ttroubleshooting, 549

overview, 534, 545pulse-chase labeling of antigens with

[35S] methionine (protocol),552–557

discussion, 555–556materials, 552–553method, 553–555monolayer-cultured cells, 554recipes, 556–557suspension-cultured cells, 554–555

radioisotopes used in metaboliclabeling, 559t

strength of antibody binding, 25ttandem immunoaffinity purification using

anti-FLAG and anti-HAantibodies (protocol), 607–612

discussion, 611materials, 607–608method: anti-FLAGpurification,607–608method: anti-HA purification, 609–610recipes, 612troubleshooting, 610t–611t

visualizing immunoprecipitated proteins,541–542

Index / 837

Inclusion bodies, 56sarkosyl preparation of antigens from

bacterial inclusion bodies(protocol), 95–96

Inclusion body lysis buffer (recipe), 96Incomplete Freund’s adjuvant, 114India ink, 81, 497Indirect ELISA

advantages and disadvantages, 633fantibody capture assay, 211fantibody capture in polyvinyl chloride wells

(protocols)enzyme-linked detection, 238–239enzyme-linked detection when immu-

nogen is an immunoglobulinfusion protein, 240–241

cell-based ELISA, 661–665, 663f–664fcompetitive, 670–673, 671f, 673fdirect versus, 632, 633fimmunometric, 644–650, 644f–645f,

647t–649tInfluenza hemagglutinin, 10fInjection routes, 120–127

anaphylactic shock and, 122boosts, 124footpad, 123, 168–169, 169fgenetic (naked DNA) immunization, 125hock, 123–124, 168–169, 169fintradermal, 121–122, 159–160, 160fintramuscular, 121, 157–158, 158fintraperitoneal, 122–123, 165–167,

165f–167fintravenous, 122, 161–164, 162f, 164finto lymphoid organs, 123–124for mice, rats, and hamsters, 120toverview, 120protocols, 150–169

footpad or hock immunization of mice,rats, and hamsters, 168–169, 169f

immunizing mice and rats withnitrocellulose-bound antigen,154–156, 155f

intradermal injection in rabbits,159–160, 160f

intramuscular injection in rabbits,157–158, 158f

intraperitoneal injection with adjuvant inmice and rats, 165, 165f

intraperitoneal injection withoutadjuvant in mice and rats,166–167, 166f–167f

intravenous injection in mice, 163–164,164f

intravenous injection in rabbits,161–162, 162f

subcutaneous injection in rabbits, 150–151subcutaneous injection with adjuvant

into mice and rats, 152subcutaneous injection without adjuvant

into mice and rats, 153for rabbits, 121trepetitive immunization at multiple sites

(RIMMS), 124, 125f, 125tsubcutaneous, 120–121, 150–153

Innate immunitydescribed, 1link to adaptive immunity, 2–3

Insect. See also Baculoviruscells, 58, 58t

Insoluble proteins as immunogen form forimmunizing animals, 117t,118–119, 118t

Institutional safety office, 819Integra CELLine 1000 flask, 314, 314fIntensifying screens, 775Interleukin-6 (IL-6), 34Intermolecular bridges, 25, 26fInternalizing antibodies, screening for, 215, 216fInterspecies hybridomas, 205, 234Intracellular staining by flow cytometry/FACS,

249–250Intradermal injection, 121–122

in rabbits (protocol), 159–160, 160fIntramuscular injection, 121

in rabbits (protocol), 157–158, 158fIntraperitoneal injection, 122–123

with adjuvant in mice and rats (protocol),165, 165f

without adjuvant in mice and rats(protocol), 166–167, 166f–167f

Intravenous injection, 122in mice (protocol), 163–164, 164fin rabbits (protocol), 161–162, 162f

InvivoGen PlasmoTest Kit, 338–339, 339fIodine

detecting iodine-labeled reagents, 727–728iodination of antibodies with immobilized

iodogen (protocol), 462–463Iodoacetyl, in conjugation of peptides to

thiol-reactive gel protocol,401–402

Ion-exchange chromatography, for cleanup andpurification of antibodyconjugates, 464–465

Ion-exchange methods, 374–375Ionic detergents, 535, 805, 806tIP. See ImmunoprecipitationIsoelectric focusing

two-dimensional isoelectric focusing/SDS-polyacrylamide gelelectrophoresis, 762–764

Isoflurane, 112, 113t, 136–137, 136f, 140Isothermal calorimetry (ITC), 361Isotype

described, 361human isotype classes and associated

Fc-binding proteins, 363tisotyping, 361–362, 366–370

Isotype determination of rodent-derived mono-clonal antibodies using sandwichELISA (protocol), 366–370

materials, 366–367method, 367–369

binding anti-light-chain detectionantibodies, 369

blocking the wells, 367capturing sample and positive control

antibodies, 369coating ELISA plate with capture

antibody, 367color development, 369plate layouts, 368fpreparing anti-light-chain detection

antibodies, 369sample preparation, 367

recipe, 370troubleshooting, 370

ITC (isothermal calorimetry), 361

Kk gene, DNA rearrangement and, 14–15, 15fKetamine/diazepam for anesthesia, 140Ketamine/xylazine for anesthesia, 113t

Keyhole limpet hemocyanin (KLH)choosing as carrier, 54coupling antigens to, 62coupling haptens to, 50cross-linking peptides to KLH with

maleimide (protocol), 85–86KLH buffer (recipe), 86Knockout mice, for antibodies against highly

homologous protein, 53Kunkel mutagenesis, 422Kymab, 236Kymouse, 236Kyte-Doolittle hydrophobicity program, 51

LLabeling antibody

cleanup and purification of antibodyconjugates, 464–468

affinity chromatography, 465desalting or buffer exchange using

size-exclusion chromatography(protocol), 467–468

dialysis, 465–466ion-exchange chromatography, 464–465overview, 464–466, 464treversed-phase chromatography, 465size-exclusion chromatography, 464,

464t, 467–468cross-linker selection, 431–433, 432t, 433t

amines/carbonyls, 431–432, 432t, 433tcarbohydrates (aldehydes), 432, 433tsulfhydryls, 432t, 433, 433t

labeling by biotinylation, 435–441labeling using biotin polyethylene oxide

(PEO) iodoacetamide (protocol),438–439

labeling using hydrazide-LC-biotin(protocol), 440–441

labeling with NHS-LC-Biotin (protocol),436–437

overview, 435labeling with fluorophores, 442–447

labeling using a maleimido dye(protocol), 446–447

labeling using NHS-fluorescein(protocol), 444–445

list of fluorophores, 442toverview, 442–443, 442t

labeling with metals and elements, 455–460labeling with colloidal gold (protocol),

459–460labeling with europium (protocol),

457–458overview, 455–456

overview, 429–430, 430fprotein–antibody conjugate formation,

448–454conjugation to horseradish peroxidase

(HRP) (protocol), 450–451labeling with Cy5-phycoerythrin

(protocol), 452–454overview, 448–449

protocols, 435–468radiolabeling, 461–463

autoradiography protocol, 83–84iodination with immobilized iodogen

(protocol), 462–463overview, 461SDS-PAGE gel and, 55

storage of antibody conjugates, 466target choice, 430–431

838 / Index

Label medium (recipe), 550, 556, 562Lactobacillus bulgaricus, 116Laemmli sample buffer, 474–475Laemmli sample buffer (2�) (recipe), 606, 612l gene, DNA rearrangements, 15, 16flgt11, 812fl repressor, affinity of, 24Large-scale antibody production, 382–383Lentivirus expression vector, 104–105Light chain, 10, 12, 12f–13f, 14Limit of detection (LoD), ELISA, 641Limit of linearity (LoL), ELISA, 640Lincomycin, addition to culture medium,

340–341Linearity, ELISA, 641Lipid A, 113Lipopolysaccharide (LPS), 113Liposomes, 113Liquid nitrogen storage

freezing cells for, 321–322recovering cells from, 323–324

Liquids, concentrations of commercial, 803tLive cells

immunogen form for immunizing animals,117t, 118t, 119

preparation for immunization(protocol), 100

LoD (limit of detection), ELISA, 641Log-odds matrix for relationships between

protein sequences, 785tLoL (limit of linearity), ELISA, 640Low-density growth supplements, 217Lowry protein quantification, 792LPS (lipopolysaccharide), 113Lymph node

harvesting, 220fharvesting from mice, rats, and hamsters

(protocol), 198–199, 199finjection into, 123purification of B cells by negative

selection, 282Lymphoid organs, injection into, 123–124Lymphokines, 113Lyophilization

for antibody storage, 383–384of gel slice (protocol), 78

Lysine. See Poly-L-lysineLysing yeast (protocol), 708Lysis. See Cell lysisLysis buffers, 534–535

recipeslysis buffer 1 (1�), 583, 587lysis buffer 2 (1�), 583lysis buffer 1 containing protease

inhibitors, 583lysis buffer 1 for ChIP, 623lysis buffer 2 for ChIP, 623lysis buffer 3 for ChIP, 623

Lysozyme, epitope of, 22Lysozyme–antibody complex, 22–23

MMacrophages, 33–34

peritoneal macrophage feeder platepreparation, 266

ridding cell lines of contaminatingmicroorganisms with peritonealmacrophages, 327

ridding cells of mycoplasma contaminationusing antibiotics and single-cell cloning (protocol), 340–341

Magic Mouse adjuvant, 115, 146–147Magnetic beads-immunoprecipitation,

540–541, 793Major histocompatibility complex (MHC), 2, 2fMajor histocompatibility complex (MHC) class

II protein, 34–36, 45alleles for, 111, 111tfailure to bind antigen fragments, 47

Major histocompatibility complex (MHC) classII-T-cell receptor binding site

coupling antigens and, 62–63synthetic, 45, 47, 47t

Major histocompatibility complex (MHC) classII-T-cell receptor sites, 63

Maleimide, 433, 433tin conjugation of peptides to thiol-reactive

gel (protocol), 401–402cross-linking peptides to KLH with

maleimide (protocol), 85–86horseradish peroxidase and, 451labeling antibodies using a maleimido dye

(protocol), 446–447Maleimide linkage, 54Maltose-binding protein (MBP), 55, 56t

as affinity tag, 539MBP-fusion protein preparation from

bacteria (protocol), 92–94Mammalian cells

mFc- and hFc-fusion protein preparation(protocol), 97–99

for recombinant protein expression, 56–57,57t, 58t

Mammalian overexpression vectors, preparingantigens from, 56–57, 57t

Mapsbacterial expression and

b-gal fusion vectors, 815fgateway expression vectors, 817flgt11, 812fpcDNA 3.1, 816fpDEST 8, 818fpDEST 15, 817fpDEST 27, 817fpET-SUMO, 816fpFastBac1, 816fplasmids, 813f–818fTrp fusion vectors, 814fT7 vectors, 813f

partial proteolytic peptide maps, 760–761Marginal ear vein, sampling rabbit serum from

(protocol), 170–171, 170fMaterial safety data sheets (MSDSs), 819Matrigel, embedding cultured cells in

(protocol), 701–702MBP. See Maltose-binding proteinMBP binding buffer (recipe), 93MBP elution buffer (recipe), 93MBP-fusion protein preparation from bacteria

(protocol), 92–94MBP lysis buffer (recipe), 94MDP (muramyl dipeptide), 113Medical Pathological Waste (MPW), 821MegAlign program, 51Membranes, transfer of proteins from gels to,

497–508overview, 497semi-dry electrophoretic transfer (protocol),

499–502, 500fstaining the blot for total protein with

Ponceau S (protocol), 507–508wet electrophoretic transfer (protocol),

503–506

Memory B cells, 3, 3f, 4, 32–33, 39–40, 40fsecondary response and, 32, 33

Memory T cells, 33Metabolic labeling

of antigens with [32P] orthophosphate(protocol), 558–562

discussion, 561materials, 558–559method, 559–561for monolayer-cultured cells, 559–560recipes, 561–562for suspension-cultured cells, 560–561

of antigens with [35S] methionine(protocol), 546–551

discussion, 549–550materials, 546–547method, 547–549recipes, 550–551suggested labeling amounts, 547ttroubleshooting, 549

radioisotopes used in, 559tMetal salts, detecting horseradish peroxidase-

labeled cells using diaminoben-zidine and (protocol), 715

Metals and elements, labeling antibodies with,455–460

labeling with colloidal gold (protocol),459–460

labeling with europium (protocol), 457–458overview, 455–456

colloidal gold, 455heavy metal chelators, 455quantum dots, 455–456

Methioninemetabolic labeling of antigens with [35S]

methionine (protocol),546–551

pulse-chase labeling of antigens with [35S]methionine (protocol), 552–557

Methotrexate, 205, 218, 316Metoclopramide, 141MFc- and hFc-fusion protein preparation

from mammalian cells(protocol), 97–99

MFc IgG2a receptor, 57MHC. See Major histocompatibility complexMice

administering anesthesia to (protocol),136–138, 137f

adoptive transfer immunization of mice(protocol), 191

age of immune maturation, 112antigen dose for, 116, 117tascites induction, 129

in BALB/c mice using myeloma cells(protocol), 183

binding characteristics of Protein A, ProteinG, and Protein L, 212t

bleed sample amount from, 110boost injections in, 124cDNA immunization (protocol), 192–193,

193fcell lines used for myeloma induction,

204–205, 204tchoice of species for producing antibodies,

110, 111–112decoy immunization for (protocol), 190footpad or hock immunization (protocol),

168–169, 169fhaplotypes, 111tIgG binding affinity for Proteins A, G, and L,

539–540, 540t

Index / 839

Mice (Continued)immunizing with nitrocellulose bound

antigen (protocol), 154–156,155f

injection routes for, 120, 120tinjection volume for, 121intraperitoneal injection

with adjuvant in (protocol), 165, 165fwithout adjuvant in (protocol),

166–167, 166f–167fintravenous injection in (protocol),

163–164, 164fkilling using carbon dioxide asphyxiation

(protocol), 194–195lymph node harvesting from (protocol),

198–199, 199fnext generation, 236repetitive immunization at multiple sites

(RIMMS) (protocol), 188ridding cell lines of contaminating

microorganisms by passagethrough mice (protocol),328–329

ridding cells of mycoplasma contaminationby passage through mice(protocol), 342–343

serum sampling protocolsfrom retro-orbital sinus, 175–176, 176ffrom saphenous vein, 179–180, 180ffrom submandibular vein, 177–178, 177ffrom tail vein, 172–173, 173f

spleen harvesting from (protocol), 196–197,197f

standard immunization (protocol), 184–185strains of laboratory, 803tsubcutaneous injection

with adjuvant into (protocol), 152without adjuvant into (protocol), 153

subtractive immunization (protocol), 189test bleeds in, 128–129

Micelle molecular weight, 804Microbial invasion, immune response to, 1–2Microorganisms, contamination of cell cultures

by, 230f, 309–310overview, 309–310ridding cell lines of contaminating

microorganismsby antibiotics (protocol), 325–326by passage through mice (protocol),

328–329with peritoneal macrophages (protocol),

327Microtiter plate

selection for ELISA, 632–635surface properties, 634twell shape and dimensions, 634f

Microwave safety, 820Mineral oil in myeloma induction, 204Mitochondrial genetic code, 782tMobility of amino acid residues, 52Modifying antigens. See Antigen modificationMolecular weight standards, 779tMonkeys

IgG binding affinity for Proteins A, G, and L,540t

Monoclonal antibodies. See also Hybridoma(s)affinity of, 24binding to multimeric antigen, 25for cell staining, 677t, 678choosing versus polyclonal, 109, 110t,

203t, 354class-switching variants, selecting, 233–234

decision to boost again or fuse, 126history of development, 202immunoblotting, 472t, 473immunochemical techniques using, 203timmunological techniques, 47tfor immunoprecipitation, 538isotyping and subclassing of, 231–234, 361,

366–370Cytometric Bead Assay (CBA), 232ELISA assays, 232Ouchterlony double-diffusion assay,

231–232polyclonal antibody versus, 47, 48producing (see Monoclonal antibody

generation)properties of, 109–110, 109tspecificity of, 48, 353–354usefulness of, 203, 203t

Monoclonal antibody generationcomparison of production methods, 313thybridomas (see Hybridoma production)protocols, 238–301

antibody capture in polyvinyl chloridewells

enzyme-linked detection (indirectELISA), 238–239

enzyme-linked detection whenimmunogen is an immunoglo-bulin fusion protein (indirectELISA to detect Ig fusion pro-teins), 240–241

antibody capture on nitrocellulosemembrane

dot blot, 242–243high-throughput western blot assay

for hybridoma screening, 244–245

antibody capture on permeabilizedwhole cells

immunofluorescence, 253–254intracellular staining by flow cytom-

etry/FACS, 249–250antibody capture on tissue sections

(immunohistochemistry),255–257

antibody capture on whole cellscell-surface binding (surface staining

by flow cytometry/FACS),246–248

cell-surface binding (surface stainingby immunofluorescence),251–252

antigen capture in solution:immunoprecipitation, 263

antigen capture in 96-well plates (captureor sandwich ELISA), 258–260

antigen capture on nitrocellulose mem-brane: reverse dot blot, 261–262

determining the class and subclass of amonoclonal antibody

by oucherterlony double-diffusionassays, 291–293, 292f

using antibody capture on antigen-coated plates, 294–296, 295f

using antibody capture on anti-immunoglobulin antibody-coated plates, 297–299

using flow cytometry, 300–301electro cell fusion, 281–285, 282ffibroblast feeder cell culture preparation,

271fusion by sendai virus, 279–280

myeloma cell feeder plate preparation,267–268

peritoneal macrophage feeder platepreparation, 266

polyethylene glycol fusion, 274–278screening for good batches of fetal bovine

serum, 264–265screening for good batches of polyethy-

lene glycol, 272–273single-cell cloning by growth in soft agar,

288–290single-cell cloning by limiting dilution,

286–287splenocyte feeder cell culture prepara-

tion, 269–270MOPC (mineral oil plasmacytoma), 204Mounting cell or tissue samples (protocols)

in DPX, 730in Gelvatol or Mowiol, 731

Mouse antibody production, 60Mowiol, mounting cell or tissue samples in

(protocol), 731MPW (Medical Pathological Waste), 821MRC-5 fibroblasts, 271MSDSs (material safety data sheets), 819Multiplexing, flow cytometry and, 737, 740Muramyl dipeptide (MDP), 113Murine IL-6, recombinant, 217Mutagenesis

for epitope mapping, 355–356Kunkel, 422modification of antibody function by

(protocol), 422–423materials, 422method, 423

Mycobacterium tuberculosis, 62, 113, 114, 142Mycoplasma contamination of cell cultures,

310–313, 311t–312tcolony appearance on agar plates, 330–331,

331fdetecting, 310–312, 311t, 330–339effects on cultured cells, 310, 311teliminating, 312–313, 340–343protocols

ridding cells of mycoplasma contamina-tion by passage through mice,342–343

ridding cells of mycoplasma contamina-tion using antibiotics and single-cell cloning, 340–341

testing for mycoplasma contaminationby growth on microbial media,330–331, 331f

agar plate method, 330–331broth culture method, 331

testing for mycoplasma contaminationby Hoechst dye 33258 staining,332–333, 333f

testing for mycoplasma contaminationusing PCR, 334–337, 335f

testing for mycoplasma contaminationusing reporter cells, 338–339,339f

resistance to antibiotics, 312, 312t, 313sources of contamination, 310, 312t

Myeloma. See also Hybridomasascites induction in BALB/c mice using

myeloma cells (protocol), 183from BALB/c mice, 204–205cell feeder plate preparation (protocol),

267–268cell line family tree, 204f

840 / Index

cell lines used as fusion parents, 204tcell repositories, 309fusion of cells

elimination of unfused cells by drugselection, 205, 206f

polyethylene glycol as fusing agent, 205preparing cells for fusions, 219, 219f

fusion protocolselectro cell fusion, 281–285fusion by Sendai virus, 279–280polyethylene glycol fusion, 274–278

growingcell culture, 304–309

antibiotics for, 304, 306tlong-term storage for, 309media, 304–307, 305t–306tsending and receiving myelomas, 309suppliers of media, 307ttechnique for, 307–308

contamination of cell cultures, 309–313by bacteria, fungi, or yeast, 309–310by mycoplasma, 309–313, 311t–312t

counting myeloma cells (protocol),318–319, 319f

protocols, 318–352selecting myeloma cells for HGPRT

mutants with 8-azaguanine(protocol), 349

NNABP-NF, detecting alkaline phosphatase-

labeled cells (protocol), 718NanoDrop protein quantification, 793Nanoparticles, gold, 455, 459–460Naphthol-AS-BI-phosphate (NABP)/New

Fuchsin, detecting alkalinephosphatase-labeled cells(protocol), 718

National Institute of Environmental Health andHuman Services, 823

Native proteins, 49t, 54–55Natural killer cells, ADCC and, 751NBT stock solution for AP (recipe), 524Neo-antigenic, 57Neonatal Fc receptor (FcRn), 236Neuraminidase, epitope of, 22Neuraminidase–antibody complex, 22–23NHS. See N-hydroxysuccinimideN-hydroxysuccinimide (NHS), 377, 431–432

cross-linking antibodies to beads and, 591,592f, 597

labeling antibodies using NHS-fluorescein(protocol), 444–445

labeling antibodies with NHS-LC-Biotin(protocol), 436–437

96-well platesantigen capture in (capture or sandwich

ELISA), 258–260described, 633–634hybridoma plating strategies

multiple cells per well, 223, 223fsingle cell per well, 222–223, 222f

staining cells for flow cytometry in, 247–248Nitrilotriacetic acid (NTA), 375Nitro blue tetrazolium/bromochloroindolyl

(NBT/BCIP) substrate forhorseradish peroxidase, 519, 522

Nitrocelluloseadjuvant, 114tantibody capture on nitrocellulose membrane

dot blot, 242–243

high-throughput western blot assay forhybridoma screening, 244–245

antigen capture on nitrocellulose membrane:reverse dot blot (protocol),261–262

blocking and incubation of immunoblotswith antibodies, 509–518

immunoblots prepared with immuno-precipitated protein antigens(immunoprecipitation/westernblotting), 515–518

immunoblots prepared with whole-celllysates and purified proteins(straight western blotting), 510–514

electrophorectic transfer to membranes(protocol), 81–82

immunizing mice and rats withnitrocellulose-bound antigen(protocol), 154–156, 155f

immunoblotting, 470staining immunoblot for total protein with

Ponceau S (protocol), 507–508transfer of proteins from gels to membranes,

497–508overview, 497semi-dry electrophoretic transfer

(protocol), 499–502, 500fstaining the blot for total protein with

Ponceau S (protocol), 507–508wet electrophoretic transfer (protocol),

503–506Noncovalent interactions, in antibody–antigen

complex, 23Non-fat dried milk, as blocking solution, 509Nonidet P-40, 535Nonidet P-40 lysis buffer (recipe), 567Nonionic detergents, 535, 805, 806tNOTA (1,4,7-triazacyclononane-

N,N0,N0 0-triacetic acid), 455N-succinimidyl S-acethylthioacetate (SATA), 433

in antibody conjugation to horseradishperoxidase protocol, 450–451

labeling antibodies using a maleimido dye(protocol), 446–447

NTA (nitrilotriacetic acid), 375Nucleic acids, as immunogen form for immu-

nizing animals, 117t, 118t, 119Nystatin, 325

OOccupational Safety and Health Administration

(OSHA), 819Oligonucleotides, creation of recombinant anti-

bodies using degenerate oligio-nucleotides (protocol), 407–409

discussion, 408–409materials, 407–408method, 408

Oncogenes, 235OPI (oxaloacetate pyruvate insulin), 217, 226,

269–270, 286–290OPI supplement (recipe), 270, 285, 287, 290Optical biosensor methods for measurement

of antibody affinity, 360–361Organic solvents, fixing attached cells in, 703–704Orthophosphate

metabolic labeling of antigens with [32P]orthophosphate (protocol),558–562

OSHA (Occupational Safety and HealthAdministration), 819

Ouchterlony double-diffusion assays, 231–232determining the class and subclass of a

monoclonal antibody (protocol),291–293, 292f

precipitin line, 232, 292fOvalbumin (OVA)

coupling antigens to, 62coupling haptens to, 50

Oxaloacetate pyruvate insulin (OPI), 217, 226,269–270, 286–290

PPAGE. See Polyacrylamide gel electrophoresisPall Corporation, 382–383Pam3Cys-Ser-(Lys)4 adjuvant, 115–116Paraffin tissue sections, preparing (protocol),

696–697Paraformaldehyde

fixing attached cells in (protocol), 705–706fixing suspension cells with (protocol), 707

Paraformaldehyde (4%) (recipe), 692, 695, 699, 706Paratopes, 354–355Partial proteolytic peptide maps, 760–761Particulate proteins, as immunogen form for

immunizing animals, 117t, 118tPasteurella infections, 114pBluescript, 412, 414PBS (recipe), 386, 394, 396, 400, 404pcDNA 3.1, 816fpComb3H, 422PCR. See Polymerase chain reactionpDEST 8, 818fpDEST 15, 817fpDEST 27, 817fPEG. See Polyethylene glycolPenicillin, addition to culture media, 304, 306t,

312, 325PEO (polyethylene oxide) iodoacetamide,

for labeling antibodies bybiotinylation, 438–439

Pepsin, 438Peptide(s). See also Synthetic peptides

conjugation of peptides to thiol-reactive gel(protocol), 401–402

designing against highly homologousextracellular proteins, 53

sequence, choosing the appropriate, 52size, 53

Peptide libraries, overlapping for determinationof epitope location, 356–357

Peptide maps, partial proteolytic, 760–761Peptide-specific antibodies, affinity purification

of, 378Peritoneal macrophages

feeder plate preparation, 266ridding cell lines of contaminating

microorganisms with peritonealmacrophages, 327

Permeabilized whole cells, antibody capture onimmunofluorescence, 253–254intracellular staining by flow cytometry/

FACS, 249–250pET-SUMO, 816fpFABC, 412, 413f, 415–416pFastBac1, 816fpH

of antigen preparation, 54cross-linking and, 62

Phagecollection of particles, 420panning for, 420

Index / 841

Phage display, creation of recombinantantibodies using (protocol),412–421

discussion, 420materials, 412–414method, 414–420

amplification of gene III, 415construction of cDNA library, 417–420,

419tconstruction of Gene III-kappa c-region

insert and ligation into pPDS,415–416

construction of pLINK and pFABC,416–417

construction of pPDS vector, 414primer list, 419t

Phagemidscommercially available vectors, 412in creation of recombinant antibodies

using phage display (protocol),412–421

Phagocytosis, 33, 34, 35fPhosphatase inhibitors, in lysing yeast cells with

glass beads protocol, 579–580Phosphate-based immunoblot wash buffer

(PBST) (recipe), 508, 513,518, 529

Phosphate-buffered saline (PBS) (10�)(recipe), 513

Phosphate buffered saline (PBS) (pH 7.4) (rec-ipe), 650, 656, 660, 664, 668, 673

Phosphatidyl serine, 749Phospho-specific antibodies, screening for,

214–215Photographing samples (protocol), 733Phycobilins, 448Phycoerythrin

antibody conjugation to, 448, 452–454properties of, 723t

Pigsbinding characteristics of Protein A, Protein

G, and Protein L, 212tIgG binding affinity for Proteins A, G, and

L, 540tPlaque assay, 102–103Plasmablasts, 3, 3f, 32, 40, 40fPlasma cells, 3, 3f, 4, 32, 39, 45, 202Plasmid maps for bacterial expression,

813f–818fPlasmoTest Kit, 338–339, 339fpLenti/-TOPO expression vector, 104–105pLINK, 412, 413f, 415–416Polyacrylamide gel electrophoresis (PAGE)

alternate buffer systems for discontinuousPAGE, 759t

protocols, 755–757alkylation of proteins before running on

a gel, 765electroelution of protein antigens from

polyacrylamide gel slices, 79–80fragmenting a wet gel slice, 77lyophilization of gel slice, 78partial proteolytic peptide maps,

760–761resolving proteins by, 490–496, 495t

gel formulations, 495tmaterials, 491–492method, 492–494overview, 490recipes, 495–496separation range, 495ttroubleshooting, 494–495

side-strip method, 76staining

Coomassie Brilliant Blue, 73copper chloride, 75sodium acetate, 74

two-dimensional isoelectric focusing/SDS-polyacrylamide gelelectrophoresis, 762–764

in recombinant protein preparationprocess, 56

solutions for, 758tPolyacrylamide gels

counting 32P or 125I from slices, 767counting 35S, 14C, 3H from slices, 767drying, 773fixing, 773rehydrating dried, 774transfer of proteins from gels to membranes,

497–508overview, 497semi-dry electrophoretic transfer

(protocol), 499–502, 500fstaining the blot for total protein with

Ponceau S (protocol), 507–508wet electrophoretic transfer (protocol),

503–506Polyclonal antibodies

animal species choice for producing, 110–111for cell staining, 677–678, 677tchoosing versus monoclonal, 109, 110t,

203t, 354by hyperimmunization, 126–127immunoblotting, 472–473, 472timmunochemical techniques using, 203timmunological techniques, 47tfor immunoprecipitation, 537–538monoclonal antibody versus, 47–48multiple epitopes recognized by, 353–354properties of, 109, 109t

Polyethylene glycol (PEG)as fusing agent in hybridoma production,

205, 222fusion (protocol), 274–278

day before fusion, 274–275materials, 274method, 274–276postfusion cell culture, 276recipes, 277–278sample preparation, 275spleen-myeloma fusion, 275–276troubleshooting, 276–277

method for isolation of IgY from chickeneggs, 399–400

screening for good batches (protocol),272–273

spacer, 436Polyethylene oxide (PEO) iodoacetamide,

for labeling antibodies bybiotinylation, 438–439

Poly-histidine tags, 89–91Poly-L-lysine

attaching suspension cells to slides using(protocols), 689

attaching yeast cells to slides using(protocol), 691–692

Polymerase chain reaction (PCR)creation of recombinant antibodies using

degenerate oligionucleotides(protocol), 407–409

creation of recombinant antibodies usingphage display (protocol),412–421

creation of recombinant antibodies using50-RACE (protocol), 410–411

MAP testing using, 60modification of antibody function by

mutagenesis (protocol), 422–423rescue strategy for nonviable hybridomas

(protocol), 424–427single-cell PCR technology, 235testing for mycoplasma contamination using

PCR (protocol), 334–337, 335fPolysorbate 80, 384Polyvinyl chloride wells, antibody capture in

enzyme-linked detection (indirect ELISA),238–239

enzyme-linked detection when immunogenis an immunoglobulin fusionprotein (indirect ELISA to detectIg fusion proteins), 240–241

Polyvinylidene fluoride (PVDF) membranesblocking and incubation of immunoblots

with antibodies, 509–518immunoblots prepared with immuno-

precipitated protein antigens(immunoprecipitation/westernblotting), 515–518

immunoblots prepared with whole-celllysates and purified proteins(straight western blotting),510–514

immunoblotting, 470staining immunoblot for total protein with

Ponceau S (protocol), 507–508transfer of proteins from gels to membranes,

497–508overview, 497semi-dry electrophoretic transfer

(protocol), 499–502, 500fstaining the blot for total protein with

Ponceau S (protocol), 507–508wet electrophoretic transfer (protocol),

503–506Ponceau Red dye, 55Ponceau S, 81

staining immunoblot for total protein with(protocol), 507–508

Ponceau S solution (recipe), 508Pooled monoclonal antibodies for cell staining,

677t, 678Porcine thyroglobulin (PTG), coupling antigens

to, 62[32P] orthophosphate

metabolic labeling of antigens with [32P]orthophosphate (protocol),558–562

Posttranslational modification, 56, 57affinity purification of modification

state–specific antibodies, 379location of, 377

PPD (purified protein derivative of tuberculin)coupling antigens to, 62

PPDS, 412, 413f, 414–416PPO fluorography, 766, 775Precipitation of antigen, 54Precipitin line, 232, 292fPrecision, ELISA, 640Primary response, 32Primers. See Polymerase chain reaction; specific

protocolsPROGEN Biotechnik, 409Programmed cell death (apoptosis) assessment

(protocol), 749–750, 750fProline exposure on protein surface, 52

842 / Index

Propidium iodide, 749–754Protean program, 51Protease and phosphatase inhibitors

(recipe), 570Protease-free strains, 56Protease inhibitors, 535, 536t, 794t

in lysing yeast cells with glass beads protocol,579–580

Proteases, 794tcell lysis and, 535unmasking hidden epitopes with, 680

Protein Ain antibody capture assays, 210–211, 212tavidity and, 27f, 28binding characteristics of, 212tchromatography, 375, 393–394cross-linking antibodies to beads,

591–592in cross-linking antibodies to beads using

dimethyl pimelimidate protocol,591, 592f, 593–596

in cross-linking antibodies to beads usingdisuccinimidyl subpimelimidateprotocol, 591, 592f, 597–600

IgG binding affinity for, 539–540, 540tin immune complex preparation for

injection protocol, 70–71immunoglobulin binding by, 375

Protein A/G agarose resin, 263Protein–antibody conjugate formation,

448–454labeling with Cy5-phycoerythrin (protocol),

452–454overview, 448–449

alkaline phosphatase, 448fluorescent proteins, 448–449horseradish peroxidase, 448

Protein carriers, coupling antigens to, 62Protein-free blocking buffers, 636Protein G

in antibody capture assays, 210–211, 212tchromatography, 375, 395–396cross-linking antibodies to beads, 591–592in cross-linking antibodies to beads using

dimethyl pimelimidate protocol,591, 592f, 593–596

IgG binding affinity for, 540, 540timmunoglobulin binding by, 375

Protein Lin antibody capture assays, 211, 212tin cross-linking antibodies to beads using

dimethyl pimelimidate protocol,591, 592f, 593–596

IgG binding affinity for, 540, 540timmunoglobulin binding by, 375

Protein quantification, 789–793bicinchoninic acid, 792Bradford, 789Bradford spot test, 790Coomassie spot test, 790Lowry, 792NanoDrop, 793UV detection, 791

protein solutions contaminated withnucleic acids, 791

pure protein solutions, 791Protein sequence(s)

log-odds matrix for relationshipsbetween, 785t

motifs, 787tProtein solutions, preparing for

immunoblotting, 484–486

Protein-specific antibodies, affinity purificationof, 378

Protein techniques, 777–797amino acids, 780tamino acid substitutions, 786tammonium sulfate saturation

tables, 778tchromogenic substrates

yielding water-insoluble products, 797tyielding water-soluble products, 797t

codon usage, 783t–784tconcentration of protein samples by

ultracentrifugation, 793David’s life chart II, 788tdialysis tubing preparation, 795genetic code, 781tlog-odds matrix for relationships between

protein sequences, 785tmagnetic beads-immunoprecipitation, 793mitochondrial genetic code, 782tmolecular weight standards, 779tprotease inhibitors, 794tproteases, 794tprotein quantification, 789–793

bicinchoninic acid, 792Bradford, 789Bradford spot test, 790Coomassie spot test, 790Lowry, 792NanoDrop, 793UV detection, 791

protein solutions contaminated withnucleic acids, 791

pure protein solutions, 791protein sequence motifs, 787tTCA precipitation

filtration, 795spotting, 796

Proteolytic peptide maps, partial, 760–761ProtScale, 51PTG (porcine thyroglobulin), coupling antigens

to, 62Pure antigens, 49t, 54–55Purification. See Antibody purificationPurification tags. See TagPurified native proteins, 49t, 54–55

QQuantification limit, ELISA, 639, 641Quantum dots, 455–456

RRabbits

administering anesthesia to (protocol), 139–141age of immune maturation, 112antigen dose for, 116–117, 117tbinding characteristics of Protein A, Protein

G, and Protein L, 212tbleed sample amount from, 110boost injections in, 124IgG binding affinity for Proteins A, G, and L,

539, 540tinjection routes for, 120, 121tinjection volume for, 121intradermal injection in (protocol),

159–160, 160fintramuscular injection in (protocol),

157–158, 158fintravenous injection in (protocol),

161–162, 162f

sampling serum from marginal ear vein(protocol), 170–171, 170f

species choice for producing polyclonalantibodies, 110–111

standard immunization (protocol), 186–187subcutaneous injection in(protocol),150–151test bleeds in, 128–129

Radiationcounting 32P or 125I from polyacrylamide gel

slices, 767counting 35S, 14C, 3H from polyacrylamide

gel slices, 767detecting iodine-labeled reagents in cell

staining protocols, 727–728safety procedures, 822waste disposal, 821, 822

Radiolabelingantibodies, 461–463

iodination with immobilized iodogen(protocol), 462–463

overview, 461autoradiography protocol, 83–84SDS-PAGE gel and, 55

Range, ELISA, 642RAS (Ribi Adjuvant System), 114t, 115, 144Rats

administering anesthesia to (protocol),136–138, 137f

age of immune maturation, 112antigen dose for, 116, 118tbinding characteristics of Protein A, Protein

G, and Protein L, 212tbleed sample amount from, 110cDNA immunization (protocol), 192–193, 193fcell lines used for myeloma induction, 204tdecoy immunization for (protocol), 190footpad or hock immunization (protocol),

168–169, 169fIgG binding affinity for Proteins A, G, and L,

540timmunizing with nitrocellulose bound antigen

(protocol), 154–156, 155finjection routes for, 120tinjection volume for, 121intraperitoneal injection

with adjuvant in (protocol), 165, 165fwithout adjuvant in (protocol),

166–167, 166f–167fkilling using carbon dioxide asphyxiation

(protocol), 194–195lymph node harvesting from (protocol),

198–199, 199frepetitive immunization at multiple sites

(RIMMS) (protocol), 188serum sampling protocols

from retro-orbital sinus, 175–176, 176ffrom submandibular vein, 177–178, 177ffrom tail vein, 174

spleen harvesting from (protocol), 196–197,197f

standard immunization (protocol), 184–185subcutaneous injection

with adjuvant into (protocol), 152without adjuvant into (protocol), 153

subtractive immunization (protocol), 189test bleeds in, 128–129

RBCs. See Red blood cellsRCSB tools, 51R&D Systems MycoProbe Kit, 334–337Recipes

acidic stripping buffer, 528AH selection medium, 351

Index / 843

Recipes (Continued)alkaline phosphatase buffer, 523, 720antigen dialysis buffer, 80antigen electrophoresis buffer, 80BCIP stock solution for AP, 523blocking buffer, 370blocking solutions, 513, 517, 529borate buffer (pH 8.0, 1�), 595borate buffer (pH 9.0, 1�), 595Bouin’s fixative, 699buffer A for cross-linking antibodies to

beads, 595, 599buffer A for dounce homogenization, 573buffer B for dounce homogenization, 573buffer C for dounce homogenization, 574buffer D for dounce homogenization, 574carbonate buffer, 650, 656, 660, 668, 673cDNA synthesis buffer, 411chasing medium, 556citrate binding buffer, 98citrate elution buffer, 99DAB substrate solution for HRP, 523developing solution (for AP), 239, 241developing solution (for Strep-AP), 259diaminobenzidine (DAB) solution, 257digitonin extraction buffer, 577dimethyl pimelimidate (1�), 596disuccinimidyl glutarate (0.5 M), 627D-10 medium, 277DNA loading buffer (6�), 622Dulbecco’s phosphate-buffered saline

(pH 7.2, 1�), 599EBC lysis buffer, 622ECF buffer (1�), 284elution buffer, 623ethanolamine (1�), 596FACS buffer, 745formaldehyde solution (11%), 623fusion selection (HAT) medium, 268GST elution buffer, pH 8.0, 88GST lysis buffer, 88guanidine-chloride stripping buffer, 529HAT fusion medium, 278, 284HAT selection medium, 351His-fusion binding buffer, pH 7.4, 90His-fusion elution buffer, pH 7.4, 90His-fusion lysis buffer, pH 7.4, 91HMT selection medium, 351homemade chemiluminescent substrate

solution for HRP: solution A, 523homemade chemiluminescent substrate

solution for HRP: solution B, 524hydroxylamine buffer, 447, 451inclusion body lysis buffer, 96KLH buffer, 86label medium, 550, 556, 562Laemmli sample buffer (2�), 606, 612lysis buffer 1 (1�), 583, 587lysis buffer 2 (1�), 583lysis buffer 1 containing protease inhibitors,

583lysis buffer 1 for ChIP, 623lysis buffer 2 for ChIP, 623lysis buffer 3 for ChIP, 623MBP binding buffer, 93MBP elution buffer, 93MBP lysis buffer, 94NBT stock solution for AP, 524Nonidet P-40 lysis buffer, 567OPI supplement, 270, 285, 287, 290paraformaldehyde (4%), 692, 695, 699, 706PBS, 386, 394, 396, 400, 404

phosphate-based immunoblot wash buffer(PBST), 508, 513, 518, 529

phosphate-buffered saline (PBS) (10�), 513phosphate buffered saline (PBS) (pH 7.4),

650, 656, 660, 664, 668, 673Ponceau S solution, 508protease and phosphatase inhibitors, 570RIPA buffer, 567, 623Sarkosyl buffer, pH 7.4, 96saturated ammonium sulfate, 386SDS-PAGE running buffer (1�), 495SDS-PAGE sample buffer (1�), 482SDS-PAGE sample buffer (2�), 483, 485, 489SDS-PAGE sample buffer (3�), 485SDS-PAGE sample buffer (6�), 486semi-dry transfer buffer (1�), 502semi-dry transfer buffer stock solution

(25�, without methanol), 502separating gel buffer (4�), 496sodium acetate, 388solution A for cross-linking antibodies to

beads, 596solution B for cross-linking antibodies to

beads, 596sorbitol buffer, 692stacking gel buffer (4�), 496starvation medium, 551, 556, 562stock buffer (4�), 577stock buffer (10�), 577stripping buffer with SDS, 530TBS (0.1 M), 386, 394, 396, 398, 404tissue lysis buffer, 570transfer buffer 1, 505transfer buffer 2, 506Tris-based immunoblot wash buffer (TBST),

508, 514, 518, 530Tris buffered saline (TBS), 650, 656, 660,

664, 668, 673Tris-buffered saline (TBS) (10�), 514, 518Tris dialysis buffer, 454Triton X-100 extraction buffer, 577Trypan Blue stain (0.4%), 574, 578, 583trypsin solution, 257Tween 40/deoxycholate extraction

buffer, 578wash buffer, 606, 612water-saturated isobutyl alcohol, 496

Recombinant antibodiescreation using degenerate oligionucleotides

(protocol), 407–409discussion, 408–409materials, 407–408method, 408

creation using phage display (protocol),412–421

discussion, 420materials, 412–414method, 414–420

amplification of gene III, 415construction of cDNA library,

417–420, 419tconstruction of Gene III-kappa c-

region insert and ligation intopPDS, 415–416

construction of pLINK and pFABC,416–417

construction of pPDS vector, 414primer list, 419t

creation using 50-RACE (protocol), 410–411materials, 410method, 410–411recipe, 411

modification of antibody function bymutagenesis (protocol), 422–423

materials, 422method, 423

rescue strategy for nonviable hybridomas(protocol), 424–427

materials, 424–425method, 425–427

Recombinant murine IL-6, 217Recombinant proteins

expression system selection, 55, 57t, 58tas immunogens, 49t, 55–59, 119

advantages and disadvantages of use, 49tfrom bacterial overexpression vectors,

55–56, 56tfrom baculovirus overexpression

vectors, 56choosing between proteins and peptides,

57–59, 57t, 58tfrom mammalian overexpression vectors,

56–57Recombination. See also DNA, rearrangements

allelic exclusion and, 17–18antibody diversity and, 17class and subclass switching, 18f, 19, 39T-cell receptor diversity and, 35

Recovering cells from liquid nitrogen storage,323–324

Red blood cells, coupling antigens to, 63, 72Regulatory T cells (Tregs), 6Rehydrating dried polyacrylamide gels, 774ReliaBLOT, 509Repetitive immunization at multiple

sites (RIMMS), 124, 125f,125t, 188

Reporter cells, testing for mycoplasmacontamination using (protocol),338–339, 339f

Reprobing membranes after immunoblotting,526–530

overview, 526stripping the blot for reprobing, 527–530

Rescue strategy for nonviable hybridomas(protocol), 424–427

materials, 424–425method, 425–427

Resonance wave grafting, 360Reticuloendothelial system, 33Retro-orbital sinus, sampling mouse and rat

serum from (protocol),175–176, 176f

Reverse dot blotantigen capture assay, 214protocol, 261–262

Reversed-phase chromatography, for cleanupand purification of antibodyconjugates, 465

Rhodaminedetecting fluorochrome-labeled reagents

in stained cells (protocol),722–724

properties of, 723tRibi Adjuvant System (RAS), 114t, 115, 144RIMMS (repetitive immunization at multiple

sites ), 124, 125f, 125t, 188RIPA buffer (recipe), 567, 623RLM-RACE, 410RNA blotting, 470RNA interference (RNAi), 474Roller bottles, growing hybridomas in, 314Routes of injection. See Injection routesRPMI 1640 medium, 304, 305t–306t

844 / Index

SSaccharomyces cerevisiae. See YeastSAF-1 adjuvant, 114Safety, 819–824

biological safety procedures, 822–823general cautions, 819–821institutional safety office, 819material safety data sheets (MSDSs), 819properties of common hazardous chemicals,

823–824radioactive safety procedures, 822waste disposal, 821

Sandwich ELISAantibody capture, 211fantigen capture assay, 214antigen capture in 96-well plates (protocol),

258–260checkerboard titration of antigen and

antibody by serial dilution, 654timmunometric antibody, 651–657, 652f,

654t, 655f–656fimmunometric double-antibody, 658–660,

658fisotype determination of rodent-derived

monoclonal antibodies usingsandwich ELISA (protocol),366–370

optimizing pairwise antibody concentrations,654–655

Saphenous vein, sampling mouse and rat serumfrom (protocol), 179–180, 180f

Sarkosyl buffer, pH 7.4 (recipe), 96Sarkosyl preparation of antigens from bacterial

inclusion bodies (protocol),95–96

SATA. See N-succinimidyl S-acethylthioacetateSaturated ammonium sulfate (recipe), 386[35S] cysteine

metabolic labeling of antigens with [35S]methionine (protocol), 546–551

SDS. See Sodium dodecyl sulfateSDS-PAGE. See Electrophoresis; Polyacrylamide

gel electrophoresisSDS-PAGE running buffer (1�) (recipe), 495SDS-PAGE sample buffer

for preparing protein solutions for immu-noblotting (protocol), 484–486

for preparing whole-cell lysates forimmunoblotting (protocol),480–483

SDS-PAGE sample buffer (1�) (recipe), 482SDS-PAGE sample buffer (2�) (recipe), 483,

485, 489SDS-PAGE sample buffer (3�) (recipe), 485SDS-PAGE sample buffer (6�) (recipe), 486SDS-polyacrylamide gel electrophoresis

(SDS-PAGE). See also Electro-phoresis; Polyacrylamide gelelectrophoresis

alkylation of proteins before running on agel, 765

alternate buffer systems for discontinuousPAGE, 759t

gel fragment processing, 55native protein isolation by, 54–55partial proteolytic peptide maps, 760–761protocol, 755–757solutions for, 758tstaining, 55two-dimensional isoelectric focusing/

SDS-polyacrylamide gelelectrophoresis, 762–764

SEAP (secreted embryonic alkalinephosphatase), 338, 339f

Secondary antibodies, reporter-labeled,636–637, 637t

Secondary lymphoid organs, 3–4Secondary response, 32, 33Secondary structure, predicting, 51Secreted embryonic alkaline phosphatase

(SEAP), 338, 339fSelf-tolerance, failure of, 46Semi-dry electrophoretic transfer (protocol),

499–502, 500fmaterials, 499–500method, 500–501recipes, 501–502schematic for, 500ftroubleshooting, 501

Semi-dry transfer buffer (1�) (recipe), 502Semi-dry transfer buffer stock solution (25�,

without methanol) (recipe), 502Sendai virus, 221, 279–280Sensitivity, ELISA, 641Separating gel buffer (4�) (recipe), 496Sephadex resins, 464, 464tSequence homologies, checking for, 51Sequence motifs, protein, 787tSerum

for ELISA blocking, 636preparation, 129

protocol, 181Serum-free medium, 304, 306–307, 307tSerum sampling

methods of blood sampling, 128tprotocols, 170–181

sampling mouse and rat serum fromretro-orbital sinus, 175–176, 176f

sampling mouse and rat serum fromsaphenous vein, 179–180, 180f

sampling mouse and rat serum fromsubmandibular vein, 177–178,177f

sampling mouse serum from tail vein,172–173, 173f

sampling rabbit serum from marginal earvein, 170–171, 170f

sampling rat serum from tail vein, 174test bleeds, 127–129

Sheepbinding characteristics of Protein A, Protein

G, and Protein L, 212tIgG binding affinity for Proteins A, G, and L,

540tShizosaccharomyces pombe. See YeastSide-strip method (protocol), 76Signal amplification scale, 478Silver staining of gels (protocols)

ammoniacal silver staining, 770neutral silver staining, 771

Simple multiplexed antibody-binding assay(protocol), 742–745, 744f, 744t

Single-cell cloningby growth in soft agar (protocol), 288–290by limiting dilution (protocol), 286–287ridding cells of mycoplasma contamination

using antibiotics and single-cellcloning (protocol), 340–341

6x His, 55, 56tSize-exclusion chromatography

for cleanup and purification of antibodyconjugates, 464, 464t, 467–468

desalting or buffer exchange using(protocol), 467–468

Slide-A-Lyzer MINI Dialysis Unit, 572SMARTer RACE, 410SMCC (succinimidyl 4-(N-maleimidomethyl)

cyclohexane-1-carboxylate), inantibody conjugation to horse-radish peroxidase protocol,450–451

[35S] methioninemetabolic labeling of antigens with [35S]

methionine (protocol), 546–551pulse-chase labeling of antigens with [35S]

methionine (protocol), 552–557Sodium acetate, 55

staining protocol, 74Sodium acetate (recipe), 388Sodium azide, 383Sodium deoxycholate (DOC), 535

in differential detergent lysis of cellularfractions protocol, 575–576

Sodium dinitrobenzene sulfonic acid(DNBS), 66

Sodium dodecyl sulfate (SDS)for cell lysis, 535in denaturing cell lysis (protocol), 588–590for denaturing proteins, 474–475screening for bacterial expression by SDS

lysis, 810Sodium metaperiodate, 432Sodium pentobarbital for anesthesia, 112Sodium sulfate method, for isolation of IgY from

chicken eggs, 397–398Soft agar

hybridoma plating strategies, 224, 224fsingle-cell cloning by growth in (protocol),

288–290Soluble proteins, as immunogen form

for immunizing animals, 117t,118, 118t

Solution A for cross-linking antibodies to beads(recipe), 596

Solution B for cross-linking antibodies to beads(recipe), 596

Solventsfixing attached cells in organic (protocol),

703–704safe handling of, 820, 823

Somatic hypermutation, 17, 18Somatic mutation, 33, 39Sorbitol buffer (recipe), 692SPDP (N-succinimidyl-3-(2-pyridyldithio)-

propionate), 433Specificity

antibody, 23, 353–354ELISA, 642

Spleencell collection from, 275harvesting, 196–197, 197f, 220finjection into, 123

Splenocytesfeeder cell culture preparation (protocol),

269–270preparing for fusions, 219–220, 219f–221fspleen-myeloma fusion protocol, 275–276

SPR (surface plasmon resonance), for measure-ment of antibody affinity, 360

Stacking gel buffer (4�) (recipe), 496Staining. See also Cell staining

cell-permanent dye, 742–743immunoblot, 497, 507–508protocols

Coomassie Brilliant Blue, 73maximal sensitivity, 769

Index / 845

Staining. See also Cell staining (Continued)quick method, 768standard method, 768

copper chloride, 75, 772immunoblot for total protein with

Ponceau S, 507–508silver staining of gels

ammoniacal silver staining, 770neutral silver staining, 771

sodium acetate, 74testing for mycoplasma contamination

by Hoechst dye 33258 staining,332–333, 333f

SDS-PAGE gels, 55for viability check with Trypan Blue, 320

Standards, molecular weight, 779tStarvation medium (recipe), 551, 556, 562Stock buffer (4�) (recipe), 577Stock buffer (10�) (recipe), 577Stock solutions, preparation of, 801t–802tStorage

of antibody, 383–384of antibody conjugates, 466of hybridomas, 309in liquid nitrogen

freezing cells for, 321–322recovering cells from, 323–324

Strepavidin, 632, 681enzyme-labeled, 516fluorochrome-labeled, 516–517

Strepavidin-alkaline phosphatase, 258–259StrepTactin, 55, 56tStreptomycin, addition to culture media, 304,

306t, 312, 325Stripping an immunoblot for reprobing,

527–530Stripping buffer with SDS (recipe), 530Subcutaneous injection, 120–121

with adjuvant into mice and rats(protocol), 152

in rabbits (protocol), 150–151without adjuvant into mice and rats

(protocol), 153Submandibular vein, sampling mouse and

rat serum from (protocol),177–178, 177f

Substrates, ELISA, 637–639chemifluorescent, 638–639chemiluminescent, 638chromogenic (colorimetric), 637–638

Subtractive immunization, 60for mice, rats, and hamsters (protocol), 189

Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), in antibody conjugationto horseradish peroxidaseprotocol, 450–451

N-succinimidyl-3-(2-pyridyldithio)-propionate(SPDP), 433

N-succinimidyl S-acethylthioacetate (SATA), 433in antibody conjugation to horseradish

peroxidase protocol, 450–451labeling antibodies using a maleimido dye

(protocol), 446–447Succininimidyl 4-formylnebzoate, 432Sulfhydryls

biotinylating antibodies using biotinpolyethylene oxide (PEO)iodoacetamide (protocol),438–439

cross-linking sites for labeling antibody,432t, 433, 433t

Supernatant collection strategies for screening,224–225

Suppressor B cells, 6Surface plasmon resonance (SPR), for measure-

ment of antibody affinity, 360Surface staining

by flow cytometry/FACS, 246–248by immunofluorescence, 251–252

Surfactants, 113Suspension cells

attaching cells to slidesusing cytocentrifuge, 688using poly-L-lysine, 689

fixing with paraformaldehyde, 707preparing for staining, 679

Synthetic peptides as immunogens, 49t, 50–54,119

advantages and disadvantages of use, 49t, 51carrier selection, 54choosing over recombinant protein, 57–59coupling strategy, 53–54designing the peptide, 51–52peptide design against highly homologous

extracellular proteins, 53peptide sequence choice, 52size of peptide, 53

TTag(s), 55, 56t, 58

antigenicity of, 58epitope, 539protocols

GST-fusion protein preparation frombacteria, 87–88

His-fusion protein preparation frombacteria, 89–91

MBP-fusion protein preparation frombacteria, 92–94

surface for transfection of cells, 59tandem affinity purification (TAP) tags, 539

Tail veinsampling mouse serum from (protocol),

172–173, 173fsampling rat serum from (protocol), 174

Tandem affinity purification (TAP) tags, 539Tandem immunoaffinity purification using

anti-FLAG and anti-HA anti-bodies (protocol), 607–612

discussion, 611materials, 607–608method: anti-FLAG purification, 607–608method: anti-HA purification, 609–610recipes, 612troubleshooting, 610t–611t

TAP (tandem affinity purification) tags, 539TBS (0.1 M) (recipe), 386, 394, 396, 398, 404TCA. See Trichloroacetic acid (TCA)

precipitationT-cell-B-cell synapse, 36–37T-cell-dependent antigens, 33T-cell-independent antigens, 34T-cell receptor (TCR)

diversity of, 35MHC peptide binding, 36structure of, 35T follicular helper cell activation, 36, 37f

T-cell receptor–MHC-class II protein-bindingsites, coupling antigens with,62–63

T cellshelper, 35, 36

memory, 33regulatory (Tregs), 6

T-cell tolerance, 38, 39, 46, 62TCR. See T-cell receptorTDM (trehalose dimycolate), 115TEMED (tetramethylethylenediamine), 491–493Terminal deoxynucleotide transferase (TdT),

410–4111,4,7,10-tetraazacyclododeane-tetraacetic acid

(DOTA), 455, 457Tetramethylbenzidine (TMB), 637–638Tetramethylethylenediamine (TEMED),

491–493Texas Red, 722–724, 723tT-flasks, growing hybridomas in, 314Thiol-reactive gel, conjugation of peptides to

(protocol), 401–402Tissue

antibody capture on tissue sections(immunohistochemistry),255–257

detergent lysis of animal tissues (protocol),568–570

harvesting protocols, 194–199killing mice, rats, and hamsters using

carbon dioxide asphyxiation,194–195

lymph node harvesting from mice, rats,and hamsters, 198–199, 199f

spleen harvesting from mice, rats, andhamsters, 196–197, 197f

lysates as immunogen source, 49t, 59–60lysis of, 536–537, 568–570mounting samples

in DPX, 730in Gelvatol or Mowiol, 731

preparing, overview of, 679–680preparing cell smears from tissue samples or

cell cultures (protocol), 700preparing frozen tissue sections (protocol),

693–695preparing paraffin tissue sections (protocol),

696–697preparing sections for staining, 679–680

Tissue culture. See also Cell culturescell lysis, 535–536collecting tissue culture supernatants

(protocol), 346detergent lysis of tissue culture cells

(protocol), 565–567storing tissue culture supernatants and

ascites (protocol), 347–348Tissue culture dishes, growing adherent cells on

(protocol), 687Tissue lysis buffer (recipe), 570TiterMax adjuvant, 114t, 115, 145TMB (tetramethylbenzidine), 637–638Tolerance, 6, 46Toll-like receptors (TLRs)

described, 2Magic Mouse adjuvant and, 115, 146simulation of B cells, 33, 34Toll-like receptor 2 (TLR2), for mycoplasma

detection, 338–339, 339fTonsil tissue, stained, 714fToxic compounds, 824Toxin, pertussis, 113Transfectants, use for immunization, 59–60Transfected dendritic cell immunizations

(protocol), 104–106Transfer buffer 1 (recipe), 505Transfer buffer 2 (recipe), 506

846 / Index

Transfer of proteins from gels to membranes,497–508

overview, 497semi-dry electrophoretic transfer (protocol),

499–502, 500fstaining the blot for total protein

with Ponceau S (protocol),507–508

wet electrophoretic transfer (protocol),503–506

Trehalose dimycolate (TDM), 1151,4,7-triazacyclononane-N,N0,N0 0-triacetic acid

(NOTA), 455Trichloroacetic acid (TCA) precipitation

filtration, 795spotting, 796

Tris-based immunoblot wash buffer (TBST)(recipe), 508, 514, 518, 530

Tris-buffered saline (TBS) (10�) (recipe), 514,518

Tris buffered saline (TBS) (recipe), 650, 656,660, 664, 668, 673

Tris dialysis buffer (recipe), 454Tris/glycine SDS-polyacrylamide gel

electrophoresisprotocol, 755–757solutions for, 758t

Tris/glycine SDS-polyacrylamide gel electro-phoresis, resolving proteins by(protocol), 490–496, 495t

Triton X-100, 535in differential detergent lysis of cellular

fractions protocol, 575–576in lysing yeast cells with glass beads using

lysis buffer 2 without detergents(protocol), 582

Triton X-100 extraction buffer (recipe), 577Trp fusion vectors, 814fTrypan Blue, 320, 580Trypan Blue stain (0.4%) (recipe), 574,

578, 583Trypsin, affinity of, 24Trypsin solution (recipe), 257TSA (tyramide signal amplification), 713T7 vectors, 813fTween 20, as blocking solution, 509Tween 40, in differential detergent lysis of

cellular fractions protocol,575–576

Tween 40/deoxycholate extraction buffer(recipe), 578

Two-dimensional isoelectric focusing/SDS-polyacrylamide gel electro-phoresis, 762–764

Tylosin, addition to culture medium, 340–341Tyramide signal amplification (TSA), 713Tyrosine, arsanilic acid coupling to, 67

UUltracentrifugation, concentration of protein

samples by, 793Ultrasonicators, 820–821UV detection for protein quantification, 791

protein solutions contaminated with nucleicacids, 791

pure protein solutions, 791

VVaccination. See also Antibody responses;

Immunizationadjuvants, 33–34tolerance induction, 38

VCS-M13 helper phage, 412, 420VDJ joining, heavy-chain, 16, 16fVECTASTAIN Kit, 255–256Velocimouse, 236VERSENE, 249Viability checks (protocol), 320Virus titer determination, 102–103V-J joining

k gene, 15, 15flambda, 15, 16f

V8 protease, 760–761

WWash buffer (recipe), 606, 612Waste, disposal of, 821, 822Water-saturated isobutyl alcohol (recipe), 496Western blot assay for hybridoma screening,

high-throughput (protocol),244–245

Western blotting. See also Immunoblottingblocking and incubation with antibodies:

immunoblots prepared withimmunoprecipitated proteinantigens (protocol), 515–518

blocking and incubation with antibodies:immunoblots prepared with

whole-cell lysates and purifiedproteins (protocol), 510–514

Wet electrophoretic transfer (protocol),503–506

materials, 503method, 503–505recipes, 505–506schematic of, 504ftroubleshooting, 505

Whole cellsantibody capture on whole cells (protocols)

cell-surface binding (surface staining byflow cytometry/FACS), 246–248

cell-surface binding (surface staining byimmunofluorescence), 251–252

preparing lysates for immunoblotting(protocol), 480–483

XX-gal, detecting b-galactosidase-labeled cells

using (protocol), 721X-ray crystallography, of epitopes, 355Xylene

mounting cell or tissue samples in DPX(protocol), 730

YYeast

attaching cells to slides using poly-L-lysine(protocol), 691–692

contamination of cell cultures, 230f,309–310

overview, 309–310ridding cell lines of by antibiotics

(protocol), 325–326ridding cell lines of by passage through

mice (protocol), 328–329ridding cell lines of with peritoneal

macrophages (protocol), 327lysing cells, 537

with glass beads (protocol), 579–581using lysis buffer 2 without deter-

gents (protocol), 582–584protocol, 708using a coffee grinder (protocol),

585–587protein expression system, 58tstaining cells, 679

Index / 847