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Index
Antibodies, 5, 10, 121animals, production in, 137, 154,
403,404combining site, 120diagnostic potential, 140digestion, 124immunization, 130
live vaccines, 131passive vaccines, 131,
infectious agents, responses to,132-134
bacteria, 133, 135fungi, 133helminths, 133S 136protozoa, 133, 135viruses, 133, 134
labeling with enzymes, 69,395-398
maternal, 141structure, 121, 123, 124
Anamnestic response, 126, 128Antigen, 7, 10, 117, 118
affinity and avidity, 5, 7, 127,128,129
affinity maturation, 126, 129antigens and antibodies, forces
between, 5,127, 129antigenic commonness, 130,
143antigenic determinant {see
Epitope)
antigenic site, 5, 11 8antigenicity, 116
improving of, 404immunogenicity, 116,403,404plastics, adsorption to, 45, 46,
50desorption, 49
purity, 46assays, effects on, 46-48size considerations, 1! 7
Assay choice, 39-44, 115Avidin/biotin systems, 399
ABC system, 399BRAB system, 399LAB system, 399
BBasic principles of ELISA,
9-44Binding ratios, 100-102, 106,
108Bound and free reactants, 10Buffers blocking, 3, 14, 60
BSA containing, 62inert proteins containing,
62reasons needed, 61Tween-20 containing, 62
Buffers coating, 3, 49carbonate/bicarbonate, 49phosphate-buffered saline (PBS),
49Buffers stopping, 3, 10, 72
415
416 Index
Capture ELISA, (see sandwichELISA)
antigen titration, 192-202antibody titration, 202-205
JgG as capture reagent, 199serum as capture reagent, 200problems ins 205
Charting methods, (see IQC,Standards and Validation)
Chessboard or checkerboardtitrations (CBT), 83-113
for direct ELISA reagents, 84-92for indirect ELISA reagents,
92-103for direct sandwich ELISA, 103for indirect sandwich ELISA, 112
Chromogens, 4, 10, 154ABTS (2,2"-azino di-
ethylbenzothiazolinesulfonic acid), 65
color change, 4, 665-AS (5-aminosalicy!ic acid), 4,
65color change 66
o-nitrophenyl-betagalactopyranoside, 4, 65
color change, 66OPD (o^o-phenylaraine-
diamine), 4, 65color change, 66
pnpp (para-nitrophenylphosphate),4,65
color change, 66TMB (tetra methyl benzidine), 4,
65color change, 66
Urea, 4, 65color change, 66
Coating, 3,45-51adsorption, passive, 3, 46-50antigen attachment, covalent, 50desorption, 49plastics, binding capacity, 3, 46time and temperature, 3,46,48
Color development, 3, 4, 66, 69, 72stopping color development, 4, 72stopping agents, 3, 4, 72
Competition ELISA, 11,27-38,definition (with respect to
inhibition ELISA), 22practical exercises, 205—231
Conformational epitope, 119Conjugates, 4, 10
availability, 68gluteraldehyde coupling, 396labels, (see Enzymes and
Antibodies labeling),alkaline phosphatase, (see
Enzymes)beta-galactosidase, (see
Enzymes)horseradish peroxidase, (see
Enzymes)urease, 71 (see Enzymes)
periodate coupling, 397Continuous epitope, 119Curve shapes, 176-180
D
Definitions, 10affinity and avidity, 120antibody combining site, 120antigen, 10antigenicity, 116antispecies antibodies, 10
Index 417
conformational epitope, 119continuous epitope, 119,discontinuous epitope, 119epitope, 118epitype, 118immunogenicity, 116linear epitope, 119monoclonal antibody (mAb), 121,
122paratope, 120polyclonal antibodies, 121
Descriptions of assays, 12—44,153-231
ELISA competition, 22, 23, 24,205-231
ELISA direct, 12, 13, 14,155-165
ELISA indirect, 14, 15, 16,165-191
ELISA sandwich 17-22, 192-205Dilutions, 4, 144
calculations of, 144-148in chessboard titrations (CBT),
83-113series, 150, 151
Direct ELISAchessboard titrations of antigen
and labeled antibody,84-92
exercises, 155-165principles, 12—14,
labeled antibody, 12-14
E
ELISA, 1exercises using nonpathogenic
materials, 153-231troubleshooting, 79-81stages, 45-82
systems, 9—44definition of components of
ELISA, 1-8, 10Enzymes, 10, 64,-72
alkaline phospliatase, 4, 70beta-galactosidase, 4, 64, 68horseradish peroxidase, 4,
70urease, 71
Enzyme stability, 72Enzyme systems, 64—68
alkaline phospliatase plus para-nitro phenylphosphate, 4,65, 66, 67,
beta-galactosidase, pluso-nitrophenyl-betagalactopyranoside, 4, 65, 66,68
horseradish peroxidase plushydrogen peroxide/ABTS, 4,65, 66,
horseradish peroxidase plushydrogen peroxide/5AS, 4,65, 66, 67
horseradish peroxidase plushydrogen peroxide/OPD, 4,64, 65, 66,
horseradish peroxidase plushydrogen peroxide/TMB, 4,65, 66,
urease, pH change andbromocresol purpteindicator, 4, 65, 66,68
Epitope, 118antigenic site, 118continuous epitope, 119discontinuous epitope, 119epitype, 119
418 Index
Establishment of control negativesera, 311
External quality assurance (EQA),329
Fab, 124Fab2, 125Fc, 125Frequency distribution (see
Statistics)
G
Gluteraldehyde, 396
H
Heavy chain (see Antibodystructure),! 21, 122
Herd immunity, 142
I
IgA,5, 124, 126,129,134,135, 136
IgD, 5, 126IgE, 5, 126, 136IgG, 5, 123, 126,128, 129, 134,
135, 136, 154IgM, 5, 124, 126, 128, 129, 134,
135,136Immunosorbents, 402, 403Immobilization of antigen (see
Practical aspects of ELISA,coating)
Incubation, 57plate rotation,
57-59advantages, 59
stationary incubation, 60timing of steps, 48
Indirect ELISA exercises,170-192
Internal Quality Control (IQC),347-394
Ion exchange chromatography, 401
K
Kits, 327-329,definition of, 327ruggedrtess reagents, 326shelf life reagents, 331, 332
Labeling antibodies with enzymes,69, 395-398,,
Light chains (see Antibodystructure)
Linear epitope, 119
M
Maternal antibody, 121, 122Microtiter plates,Molarities, 151-152Monoclonal antibodies (mAbs), 19,
129,233—299availability, 234comparison to polyclonal
antibodies, 129examples of use of mAbs in
ELISA, 256, 257,268-298
isotypes, 236isotyping, 260-262purification, 257, 259—261screening for activity, 235,
249-251competition ELISA, 237-249indirect ELISA, 252-255
stability, 258
Index 419
systems for using mAbs,264-268
direct ELISA, 265indirect ELISA, 265sandwich ELISA direct. 266sandwich ELISA indirect, 267
N
Negative sera and control sera,Nonspecific binding, 10, 14, 46, 50
O
Overview of ELISA, 1-8scientific disciplines needed, 1--8
P
Papain digestion, 124Passive adsorption, 10, 45, 48, 49,
50Protein A, 401Protein G, 402Pepsin digestion, 125Periodate coupling, 397Pipets, 148
calibrationmultichannel, 148,pipetting exercise, 149single channel, 149tips, 148use, 148
Plates, 3, 45, 57-59Practical aspects of ELISA, 9-44,
153-231exercises, 153-231
direct ELISA, 155-165indirect ELISA optimization.,
165-170indirect ELISA titration
antibodies, 170-181
indirect ELISA single dilutionsof test samples,182-191
sandwich (capture) ELISA totitrate antigen, 192-202
sandwich (capture) ELISA totitrate antibodies,202-205
competitive and inhibitionELISA 205-231
Prevalence (see Validation)Principles of ELISA, 9-44Production of antisera,
403^05Problems, 74-79
conjugates, 78glassware, 75incubation, 77pipets, 76,plates, 76,reading, 78, 79,sample addition, 78stopping,techniques, 74timing, 77tips, 76troughs, 76troubleshooting guide, 80, 81water, 75
Protein A, 402Protein G, 402
QQuiz on ELISA, 407-^13Quality assurance (QA),
329external quality assurance (EQA),
329Quality control <QC), 329
420 Index
internal quality control (IQC),329, 347-394
charting methods, 347-394
R
Random testing, 310Reading, 3, 10,72-74
by eye, 72, 73spectrophotometer, 45, 73,74wavelengths for, 66
ROC analysis (see statistics)
S
Salt fractionation, 400of immunoglobulins, 400
Sandwich ELISA, 11, 17-22,192-205
competition assays for sandwichELISA, 22-44, 206, 207
direct sandwich ELISA,17-19,192-201
indirect sandwich ELISA, 19-22,202-205
Shelf life reagents, 331, 332Standardization (see Validation)
calibration standards, 324in-house reference materials, 330international standards, 330working standards, 330
Solid phase, 10,45microtiter plates, 45, 46
Substrates, 77, 154reagents, (see Chromogens)
Statistics, 6, 335-345, 356-358basic terms, 6, 336, 356charting methods for IQC (see
IQC)frequency distributions,
185-189
confidence intervals, 342normal distribution, 6,187,
338-340mean, median and mode, 6.
184,338,339populations, 6, 185-189,310,336standard deviation (SD), 6, 55,
184, 185,356ROC analysis, 317-320variation, 6, 340
TTest questions on ELISA, 407-413
Test answers, 408^413Temperature, 3, 46, 48,49, 57, 77Theoretical considerations, 115-152Timing of steps, 3, 48Tips for pipets, 3, 56, 148Titration of reagents, (see
Chessboard titrations)Troughs (reservoirs), 76
U
Units, 144volumes, 144weights, 144
V
Validation of tests using ELISA,301-345
accuracy, 322, 324, 325analytical sensitivity, 305, 322,
323analytical specificity, 305, 322diagnostic sensitivity, 307, 313,
316diagnostic specificity, 307, 313,
316gold standards, 308-310
Index 421
feasibility studies, 302 gravity-feed methods, 52precision, 322, 323, special handwashing devices,ROC analysis, 317-320 52repeatability, 305, 31 ] specialist machines, 52ruggedness testing, 326 wash bottles plus multiple
selection of negative/positive delivery nozzles,thresholds, 311 51,52
Volume, units of, 144, 145 Water, 75w quality, effect on ELISA, 75
Weights, units of, 144,145Washing, 3, 10 what is known already when setting
dipping methods, 51 u p ELISA, 39, 115