04pie119 elisabro 1 lores - cultek · d-salt™ dextran column. ... reductive amination. periodate...

40

ANTIBODY LABELING

800-874-3723 • 815-968-0747 • www.piercenet.com

Chemical cross-linking reagents have become an invaluabletool in the scientific community. These reagents are used inpreparing antibody-enzyme conjugates and other labeled proteinreagents. After the protein is conjugated to an appropriateenzyme, it may then be used as a detection reagent in a varietyof assays and applications. A number of cross-linking methodshave been used to prepare enzyme conjugates. For example,an N -hydroxysuccinimide ester can be prepared from a ligandof interest, then reacted with a primary amine on the surface ofthe enzyme. While this method is necessary in some applications,such as those in which the ligand does not contain a primaryamine, it is not useful as a general-purpose method.

Antibody-Modification SitesAntibodies can be easily modified to contain labels such asbiotin, fluorescent tags or enzymes to create reagents forELISA, Western blotting, immunohistochemical staining andin vivo targeting. Pierce offers tools for a variety of antibody-modification strategies.

Understanding the functional groups available on an antibodyis the key to choosing the proper method for modification.

For example:

Primary amines (–NH2) are found on lysine residues and theN-terminus. These are abundant and distributed over theentire antibody.

Sulfhydryl groups (–SH) are found on cysteine residues andare formed by selectively reducing disulfide bonds in the hingeregion of the antibody.

Carbohydrate residues containing cis-diols can be oxidized(–CHO) to create active aldehydes. These are localized to the Fc region on antibodies and are more abundant on polyclonal antibodies.

Antigen-bindingsite Light Chains

Carbohydrate Carbohydrate

HingeRegion

CH2

CH3

CH2

CH3

CH1 CH1

VL

CL

VL

CLVH

S-SS-S

S-SS-SS-S

S-SS-S

S-S

S-SS-S

S-S

S-S

Fab (Fab')2

FC

SS

SS

SS

SS

VH

NH2

Amines onlysine residues

Sulfhydryls created whenantibody is reduced

NH2

NH2

NH2

Heavy Chains

Step 1. Preparation of Protein

For salt-free lyophilized protein:Dissolve in borate buffer.

Reconstitute fluorescent dye with DMF.

Add dye to the protein solution.Incubate for 1 hour.

Step 2. Labeling Reaction Step 3. Removal of Excess Fluorescent Dye

For proteins in buffers or salt solutions:a.) Sample volume of 100 µl or less: Exchange into borate buffer using Slide-A-Lyzer® MINI Dialysis Unit.

b.) Sample volume greater than 100 µl: Exchange into borate buffer using a D-Salt™ Dextran Column.

For sample volume of 100 µl or less:Exchange into PBS buffer using Slide-A-Lyzer® MINI Dialysis Unit.

For sample volume greater than 100 µl:Exchange into PBS buffer using a D-Salt™ Dextran Column.

EZ-Label™ Fluorescent Labeling KitsMake your own fluorescent-labeled antibody in less than two hours!

EZ-Label™ Kits are designed for labeling any size protein –small or large – even if you have only a small amount of yourprotein. Protein sample volumes ranging from 50 µl-1 ml canbe used, with protein concentration up to 10 mg/ml for eachreaction. EZ-Label™ Kits were specially developed and optimizedfor the most efficient labeling.

EZ-Label™ Kits contain everything you need to successfullylabel your antibody or protein:• Fluorescent dye provided in individual microtubes, eliminating

the need to weigh dye • Conveniently packaged dimethylformamide (DMF) to prepare

the fluorescent dye solution• Pre-made borate and phosphate buffers – just add water to

the powder and they are ready-to-use• Pre-packed, ready-to-use desalting columns for fast buffer

exchange when your protein sample volume is greaterthan 100 µl

• Slide-A-Lyzer® MINI Dialysis Units* for easy buffer exchangewhen your protein sample volume is less than or equal to 100 µl

• Amber reaction tubes – no handling in the dark required

Excitation EmissionEZ-Label™ Kit Wavelength (nm) Wavelength (nm)Fluorescein Protein Labeling Kit 491 518Rhodamine Protein Labeling Kit 544 576 Fluorescein Isothiocyanate 494 520(FITC) Protein Labeling KitThese kits contain sufficient reagents to perform five fluorescent labeling reactions,which use up to 10 mg/ml of protein for each reaction (50 µl-1 ml volume of protein).

Ordering InformationU.S.

Product # Description Pkg. Size Price53000 EZ-Label™ Fluorescein Protein Labeling Kit Kit $237

Sufficient for five coupling reactions.Includes: No-Weigh™ 6 x 1 mg

Pre-Measured Fluorescein Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

53002 EZ-Label™ Rhodamine Protein Labeling Kit Kit $237Sufficient for five coupling reactions.Includes: No-Weigh™ 6 x 0.5 mg

Pre-Measured Rhodamine Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

53004 EZ-Label™ Fluorescein Isothiocyanate Kit $237(FITC) Protein Labeling KitSufficient for five coupling reactions.Includes: No-Weigh™ 6 x 1 mg

Pre-Measured FITC Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

* Slide-A-Lyzer® MINI Dialysis Unit Technology is protected by U.S. patent # 6,039,871.

The fluorescent-labeling procedure is easy when you use EZ-Label™ Kits.

41ANTIBODY LABELING

Fluorescent Labeling

800-874-3723 • 815-968-0747 • www.piercenet.com42

ANTIBODY LABELINGEnzyme Labeling

Maleimide ActivationThe heterobifunctional cross-linker SMCC (Product # 22360)and its water-soluble analog Sulfo-SMCC (Product # 22322)have good general utility in preparing immunologically activehorseradish peroxidase or alkaline phosphatase conjugates.They are most useful when preparing conjugates of reducedIgG and F(ab′)2, because these methods involve the initial stepof preparing a maleimide-activated (sulfhydryl-reactive) enzymederivative. Studies have shown that the two-step maleimidemethod is superior to glutaraldehyde or meta-periodate methodsfor enzyme conjugation (Figure 15). The maleimide method giveshigher yields with less polymerization, producing a conjugatepreparation with superior immunoassay characteristics.

Maleimide-activated enzymes can be prepared using theheterobifunctional cross-linker Sulfo-SMCC. This reagent containsan N-hydroxy-sulfosuccinimide (Sulfo-NHS) functional groupand a maleimide functional group and it is water-soluble dueto the presence of the sulfonate (–SO3

-) group on the N-hydrox-ysuccinimide ring. The sulfonate group also contributes to thestability of the molecule in aqueous solution. A study of thehydrolysis rate of the maleimide functional group from Sulfo-SMCCshowed that it is less prone to hydrolysis to the maleamic acidthan the non-sulfonated SMCC. The maleimide groups ofSulfo-SMCC exhibit no decomposition at pH 7 at 30˚C within6 hours. The Sulfo-NHS ester group reacts with primary amineson the enzyme surface to form a stable amide bond. After thisfirst step of conjugation, the enzyme will have maleimide groupson its surface that react optimally toward sulfhydryl groupsbetween pH 6.5 and 7.5 to form stable thioether bonds. Maleimide-mediated conjugation strategies are summarized in Figure 15.

SHProtein

Protein Protein

Three methods for free sulfhydryl generation Maleimide activation of enzyme

Native protein has a freesulfhydryl on its surface.

Native protein is reacted with SATA. Blocked sulfhydryl groups are introduced on primary amines.Hydroxylamine (H) treatment generates free sulfhydryls.

Native protein contains disulfide bonds that can be reduced to generate free sulfhydryls. Enzyme is SMCC-labeled through primary amines to generate amaleimide-activated enzyme for conjugation to free sulfhydryls.

O

C

S S

MEAEDTA

SH

NH2Protein SATA

Protein

Protein

CH2 S

O

C CH3H

CH2 SH

SHProtein

Protein

H

NO

C

H

N

O

O

OO

OO

N

SMCC

NHS

OH

O

O

N

N+ H2N

S

O

ON

O N E

E

O

ON

O N

SH

E

1

2

3

Figure 15. Three strategies for maleimide-mediated conjugation of enzymes.

ANTIBODY LABELING 43

Two reagents, Mercaptoethylamine•HCl (Product # 20408)and SATA (Product # 26102), are available to produce freesulfhydryls on macromolecules for conjugation to themaleimide-activated enzymes. For labeling antibody molecules,mild reduction with Mercaptoethylamine•HCl (MEA) results intwo half-antibody fragments containing free sulfhydryl groupsin the hinge region. Labeling in this area is advantageous becauseit directs the modification away from the antigen-binding region.Native proteins lacking a free sulfhydryl on their surface canbe reacted with SATA to generate blocked sulfhydryl groups.The SATA molecule reacts with primary amines via its NHS esterend to form stable amide linkages. The acetylated sulfhydrylgroup (blocked) is stable until treated with hydroxylamine togenerate the free sulfhydryls.

Pierce offers stable, preactivated enzyme derivatives that arereactive toward sulfhydryl (–SH) groups, EZ-Link™ MaleimideActivated Alkaline Phosphatase (Product # 31486) and HorseradishPeroxidase (Product # 31485). These products eliminate thefirst step of the two-step maleimide method, simplifying andfacilitating the conjugation protocol, while saving several hours.They can be used to prepare enzyme conjugates directly fromproteins, peptides or other ligands that contain a free –SH group.Two reagents, SATA and mercaptoethylamine•HCl, are alsoincluded in the kit formats to produce free sulfhydryls onmacromolecules for conjugation.

EZ-Link® Maleimide Activated Peroxidase ReferencesChoi, J.Y., et al. (2002). J. Biol. Chem. 277, 21630-21638. Seo, Y.R., et al. (2002). Proc. Natl. Acad. Sci. 99, 14548-14553. Yoo, J.H., et al. (2004). J. Biol. Chem. 279, 848-858.


Product # Description Pkg. Size Price31486 EZ-Link® Maleimide Activated 2 mg $174

Alkaline Phosphatase31493 EZ-Link® Maleimide Activated Kit $359

Alkaline Phosphatase KitIncludes: EZ-Link® Maleimide 2 mg

Activated Alkaline PhosphataseActivation/Conjugation Buffer 20 mlBupH™ Tris Buffered Saline Pack 2 packsBupH™ Phosphate Buffered Saline Pack 1 packPolyacrylamide Desalting Column 1 x 10 mlMercaptoethylamine•HCl 6 mgSATA 2 mgHydroxylamine 5 mgDMF 1 mlColumn Extender

31485 EZ-Link® Maleimide Activated 5 mg $132Horseradish Peroxidase

31494 EZ-Link® Maleimide Activated Kit $317Horseradish Peroxidase KitIncludes: EZ-Link® Maleimide 5 mg

Activated Horseradish PeroxidaseActivated Horseradish Peroxidase 20 mlConjugation Buffer

2-Mercaptoethylamine•HCl 6 mgSATA 2 mgDimethylformamide 1 mlHydroxylamine•HCl 5 mgPolyacrylamide Desalting Column 1 x 10 ml

23460 Protein-Coupling Handle Addition Kit Kit $181Includes: SATA 2 mg

Hydroxylamine•HCl 5 mgConjugation Buffer Stock (10X) 20 mlBupH™ Pack PBS 1 packDimethylformamide (DMF) 1 mlD-Salt™ Dextran Desalting Column 1 x 5 mlColumn Extender 1Ellman’s Reagent (DTNB) 2 mgCysteine•HCl H2O 20 mg

44

ANTIBODY LABELING

Figure 16. Conjugation scheme for periodate oxidation and subsequentreductive amination.

PeriodateGlycoproteins such as horseradish peroxidase and glucoseoxidase and most antibody molecules can be activated forconjugation by treatment with periodate. Oxidizing polysac-charide residues in a glycoprotein with sodium periodate providesa mild and efficient way of generating reactive aldehyde groupsfor subsequent conjugation with amine- or hydrazide-containingmolecules via reductive amination (Figure 16). Some selectivityof monosaccharide oxidation may be accomplished by regulatingthe concentration of periodate in the reaction medium. In thepresence of 1 mM sodium periodate, sialic acid groups arespecifically oxidized at adjacent hydroxyl residues, cleaving offtwo molecules of formaldehyde and leaving one aldehyde group.At higher concentrations of sodium periodate (10 mM or greater),other sugar residues will be oxidized at points where adjacentcarbon atoms contain hydroxyl groups. This reaction shouldbe performed in the dark to prevent periodate breakdown andfor a limited period of time (15-30 minutes) to avoid loss ofenzymatic activity.

Cross-linking with an amine-containing protein takes placeunder alkaline pH conditions through the formation of Schiffbase intermediates. These relatively labile intermediates canbe stabilized by reduction to a secondary amine linkage withsodium cyanoborohydride. Reductive amination has been done

using sodium borohydride or sodium cyanoborohydride; however,cyanoborohydride is the better choice because it is more specificfor reducing Schiff bases and will not reduce aldehydes. Smallblocking agents such as lysine, glycine, ethanolamine or Tris canbe added after conjugation to quench any unreacted aldehydesites. Ethanolamine and Tris are the best choices for blockingagents because they contain hydrophilic hydroxyl groups withno charged functional groups.

The pH of the reductive amination reaction can be controlledto affect the efficiency of the cross-linking process and thesize of the resultant antibody-enzyme complexes formed. Atphysiological pH, the initial Schiff base formation is less efficientand conjugates of lower molecular weight result. At more alkalinepH (i.e., pH 9-10), Schiff base formation occurs rapidly and withhigh efficiency, resulting in conjugates of higher molecularweight and greater incorporation of enzyme when oxidizedenzyme is reacted in excess. Low molecular weight conjugatesmay be more optimal for immunohistochemical staining orblotting techniques in which penetration of the complex throughmembrane barriers is an important consideration. Washing stepsalso more effectively remove excess reagent if the conjugate isof low molecular weight, thus maintaining low background inan assay. By contrast, conjugates of high molecular weight aremore appropriate for ELISA procedures in a microplate format,where high sensitivity is important and washing off excessconjugate is not a problem.

GlutaraldehydeAnother method for conjugation uses glutaraldehyde, one of theoldest homobifunctional cross-linking reagents used for proteinconjugation. It reacts with amine groups to create cross-links byone of several routes. Under reducing conditions, the aldehydeson both ends of glutaraldehyde will couple with amines to formsecondary amine linkages. The reagent is highly efficient at proteinconjugation but has a tendency to form various high-molecularweight polymers, making results difficult to reproduce.

EZ-Link® Activated Peroxidase ReferencesSandt, C.H. and Hill, C.W. (2001). Infect. Immun. 69, 7293-7303. Turpin, E.A., et al. (2003). J. Clin. Microbiol. 41, 3579-3583.

EZ-Link® Plus Activated Peroxidase ReferencesGlover, L. (2002). Eur. J. Biochem. 269, 4607-4616. Nawa, M., et al. (2000). Clin. Diagn. Lab. Immunol. 7, 774-777. Völkel, T., et al. (2001). Protein Eng. 14, 815-823.

OHHO

RO

HOO

O

OO

RO

HOO

O

NNH2

NaCNBH3

O

ROHO

O

O

Enzyme containingpolysaccharide chains

Enzyme withreactive aldehyde groups

Reductive amination formsstable secondary amine linkage

Antibody moleculecontaining amine groups

NalO4

(oxidation)E E

E


45ANTIBODY LABELING

Alkaline PhosphataseA highly sensitive enzyme for ELISA and immunohistochemical applications.

Highlights:• Purified form – ready to conjugate without prior dialysis• Activity is not affected by exposure to antibacterial agents

such as sodium azide or thimerosal• Specific activity > 2,000 units/mg• One unit is defined as the amount that will hydrolyze

1.0 µmole of p -nitrophenyl phosphate per minute at 37°C in 1.0 M diethanolamine, 0.5 mM MgCl2, pH 7.8

Specific Activity per mg ProteinBuffer 25°C 37°C0.1 M Glycine, 1.0 mM ZnCl2, 1.0 mM MgCl2, > 500 > 1,0006.0 mM p -Nitrophenyl phosphate, pH 10.41.0 M Diethanolamine, 0.5 mM MgCl2, > 1,000 > 2,00015 mM p -Nitrophenyl phosphate, pH 9.8

ReferencesBulman, A.S. and Heyderman, E. (1981). J. Clin. Pathol. 34, 1349-1351.Cordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229.Yolken, R.H. (1982). Rev. Infect. Dis. 4, 35-68.


Product # Description Pkg. Size Price31391 ImmunoPure® Alkaline Phosphatase 20 mg $ 652

Calf intestinal. Supplied in Tris Buffer, pH ~7Triethanolamine, 1 mM MgCl2, 3 M NaCl, pH 7.6

31392 ImmunoPure® Alkaline Phosphatase 100 mg $2,528

Horseradish PeroxidaseHigh-specific enzyme activity makes it the enzyme of choice.

Highlights:• Superior to alkaline phosphatase and β-galactosidase conjugates

due to the higher specific enzyme activity• Small size (40 kDa) allows excellent cellular penetration• Variety of substrates available• Ideal in blotting and cytochemistry applications• Used as the reporter enzyme for SuperSignal®

Chemiluminescent Substrates

ReferencesCordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229.Hosoda, H., et al. (1987). Chem. Pharm. Bull. 35, 3336-3342.Passey, R.B., et al. (1977). Clin. Chem. 23(1), 131-139.Porstmann, B., et al. (1985). J. Immunol. Methods. 79, 27-37.Samoszuk, M.K., et al. (1989). Antibody, Immunoconjugates andRadiopharmaceuticals 2, 37-46.Wordinger, R.J., et al. (1987). Manual of Immunoperoxidase Techniques, 2nd Edition.Chicago: American Society of Clinical Pathologists Press, pp. 23-24.Yolken, R.H. (1982). Rev. Infect. Dis. 4(1), 35-68.


Product # Description Pkg. Size Price31490 ImmunoPure® Horseradish Peroxidase 10 mg $ 5331491 ImmunoPure® Horseradish Peroxidase 100 mg $190

U.S.Product # Description Pkg. Size Price31487 EZ-Link® Plus Activated Peroxidase 1 mg $ 63

(Periodate Activated)31488 EZ-Link® Plus Activated Peroxidase 5 x 1 mg $232

(Periodate Activated)31489 EZ-Link® Plus Activated Peroxidase Kit Kit $317

(Periodate Activated)Includes: EZ-Link® Plus Activated Peroxidase 5 x 1 mg

Sodium Cyanoborohydride Solution 1 x 0.5 mlQuenching Buffer 25 mlBupH™ Phosphate Buffered Saline Pack 500 mlBupH™ Carbonate Buffer Pack 500 ml

U.S.Product # Description Pkg. Size Price31496 EZ-Link® Activated Peroxidase 1 mg $ 58

(Glutaraldehyde Activated)31495 EZ-Link® Activated Peroxidase 5 mg $160

(Glutaraldehyde Activated)31497 EZ-Link® Activated Peroxidase Kit $317

Antibody Labeling Kit(Glutaraldehyde Activated)Includes: EZ-Link® Activated Peroxidase 5 mg

Conjugation Buffer 50 mlLysine 250 mgImmobilized Protein A/G Column 0.5 mlGentle Ag/Ab Binding Buffer 200 mlGentle Ag/Ab Elution Buffer 200 ml

Ordering Information

Pierce offers an extensive line of substrates for AP and HRP,depending upon the plate-reading equipment available and thelevel of sensitivity required in the ELISA. Chemiluminescentand chemifluorescent substrates provide a stronger signalthan colorimetric substrates, thus providing greater sensitivity.

ELISA conditions must always be re-optimized when switchingto a more sensitive substrate (Table 7). Greater dilutions of anHRP-conjugate should be used with a chemiluminescentsubstrate than with a colorimetric substrate.

46

CHOOSING A SUBSTRATE


Table 7. Properties of ELISA substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP).

Dilution range of Ab ApproximateSubstrate Product # Page Measurement - Color (From 1 mg/ml stock) Sensitivity* EnzymeSuperSignal® ELISA Femto 37075 50 425 nm chemiluminescent 1° 1:10K – 1:20K 0.17 pg/well HRP

2° 1:50K – 1:100KSuperSignal® ELISA Pico 37070 49 425 nm chemiluminescent 1° 1:1K 0.5 pg/well HRP

2° 1:10KQuantaBlu™ Substrate 15169 51 325 nm/420 nm chemifluorescent 1° 1:10K – 1:20K 0.5 pg/well HRP

2° 1:50K – 1:100K1-Step™ Ultra TMB 34028 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 2 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ Turbo TMB 34022 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 7 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ Slow TMB 34024 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 8 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100KTMB Substrate Kit 34021 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 5.5 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ ABTS 37615 52 410 nm/650 nm – Green 1° 1:500 – 1:1K 0.25 ng/well HRP

2° 1:4K – 1:50KABTS 34026 52 410 nm/650 nm – Green 1° 1:500 – 1:1K 0.25 ng/well HRP

2° 1:4K – 1:50KOPD Powder 34005 52 490 nm stopped – Green 1° 1:500 – 1:1K 7 pg/well HRP

450 nm nonstop – Yellow-Orange 2° 1:4K – 1:100KOPD Tablets 34006 52 490 nm stopped – Green 1° 1:500 – 1:1K 7 pg/well HRP

450 nm nonstop – Yellow-Orange 2° 1:4K – 1:100K1-Step™ PNPP 37621 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Kit 37620 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Tablets 34047 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Powder 34045 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5K* Actual sensitivity is unique to each antibody-antigen pair. The approximate sensitivities listed are conservative amounts that should be easily detectable for most antigens.

Horseradish Peroxidase Substrates

Horseradish peroxidase is a 40 kDa protein that catalyzesthe oxidation of substrates by hydrogen peroxide, resulting ina colored or fluorescent product or the release of light as abyproduct. HRP functions optimally at a near-neutral pH andcan be inhibited by cyanides, sulfides and azides. Antibody-HRPconjugates are superior to antibody-AP conjugates with respectto the specific activities of both the enzyme and antibody. Inaddition, a high turnover rate, stability, low cost and wideavailability of substrates make HRP the enzyme of choicefor most applications.

When selecting a substrate for HRP-based ELISAs, there are anumber of possibilities. The question of sensitivity is often theoverriding factor in making a selection. However, considerationshould also be given to whether the substrate contains harmfulsolvents, what detection equipment is available and how theassay will be measured (kinetic or end-point). In many ELISAapplications, colorimetric substrates provide a sufficient levelof sensitivity and dynamic range. This is evident with the PierceEndogen Cytokine ELISA Kits, in which a TMB substrate coupledwith a streptavidin-HRP detection system results in pg/mlsensitivity. Detection below this level requires a fluorescent orchemiluminescent signal. Characteristics of the HRP ELISAsubstrates offered by Pierce are summarized in Table 7.

Chromogenic ELISA substrates result in a soluble, coloredproduct. These substrates are used in most ELISAs becausedetection of a colored product can be performed on a spec-trophotometric plate reader. TMB (3,3′,5,5′-tetramethylbenzidine)is the most common chromogenic substrate for HRP and isavailable in several formats. 1-Step™ Ultra TMB (Product #34028) yields the greatest sensitivity among the TMB substrates,followed by 1-Step™ Turbo TMB (Product # 34022) and 1-Step™

Slow TMB (Product # 34024). The 1-Step™ Substrates arepreformulated so no mixing or pre-filtering is required. Althoughthe sensitivity is lower, the 1-Step™ Slow TMB and 1-Step™ ABTS(2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammoniumsalt) are ideal for kinetic readings. OPD (o -phenylenediaminedihydrochloride, Product # 34005) is another relatively sensitiveHRP substrate that produces a yellow-orange color.

The greatest sensitivity in ELISA applications is obtainedusing chemiluminescent or chemifluorescent substrates. Thesesubstrates have been steadily gaining in popularity becauseof their sensitivity (less than 1 pg/ml), large linear range fordetection and excellent antibody conservation. Pierce offersthe chemiluminescent SuperSignal® ELISA Pico (Product #37070) and SuperSignal® ELISA Femto (Product # 37075)Substrates and the chemifluorescent QuantaBlu™ Substrate(Product # 15169).

Enzyme-labeled reagents are detected using chromogenic,chemiluminescent or chemifluorescent substrates. Chromogenicsubstrates are generally the least expensive, while luminescentor fluorescent substrates are often more sensitive. Whenperforming ELISAs, a soluble substrate is used and converted

to a soluble end product. ELISAs are performed on polystyreneplates, and the enzyme levels are determined by monitoringsignal development with a spectrophotometer (a luminometerfor luminescence or a fluorometer for fluorescence).

Choosing a Detection Signal TypeColorimetric Substrates Chemifluorescent Substrates Chemiluminescent Substrates

ELISA • Medium/low sensitivity • High sensitivity • High sensitivity• Generally less expensive • Generally more expensive • Generally more expensive• Many substrates available • Few substrates available • Few substrates available• Slow signal generation • Rapid signal generation • Rapid signal generation• Enzyme catalyzed quickly • Enzyme activity maintained • Enzyme catalyzed quickly• Small linear range/poor low-end linearity • Large linear range/enhanced low-end linearity • Large linear range/enhanced low-end linearity• Flexible (stopped, nonstopped and • Flexible (stopped, nonstopped and • Nonflexiblekinetic assays) kinetic assays)

Detection Spectrophotometer provides Fluorometer provides Luminometer provides Equipment quantifiable results quantifiable results quantifiable results

47CHOOSING A SUBSTRATE

Figure 17. Comparison of QuantaBlu™ Substrate to other substrates.QuantaBlu™ Substrate and the colorimetric substrates were incubated for30 minutes at RT, followed by addition of a stop solution. QuantaBlu™

Substrate gave the greatest signal:noise (S/N) ratios and exhibited thelowest detection limit.

When energy in the form of light is released from a substancebecause of a chemical reaction, the process is called chemilumines-cence. Luminol is one of the most widely used chemiluminescentreagents and its oxidation by peroxide results in creation of anexcited state product called 3-aminophthalate. This product decaysto a lower energy state by releasing photons of light (Figure 18).

Figure 18. Luminol is oxidized in the presence of horseradish peroxidase andhydrogen peroxide to form an excited state product (3-aminophthalate).The 3-aminophthalate emits light at 425 nm as it decays to the ground state.

Chemiluminescent substrates have steadily gained in popularitythroughout the past decade because they offer several advantagesover other detection methods. These advantages have allowedchemiluminescence to become the detection method of choicein most protein laboratories. Using chemiluminescence allowsmultiple exposures to be performed to obtain the best image.A large linear response range allows detection and quantitation

over a large range of protein concentrations. Most importantly,chemiluminescence yields the greatest sensitivity of any availabledetection method. Using HRP as the enzyme label and SuperSignal®

ELISA Femto Chemiluminescent Substrate (Product # 37075),lower detection limits in the upper femtogram range are possiblebecause the enhancers in this substrate greatly intensify theemitted light and extend the signal duration.

Chemiluminescent substrates differ from other substrates inthat the light detected is a transient product of the reaction thatis only present while the enzyme-substrate reaction is occurring.This is in contrast to substrates that produce a stable, coloredproduct; these colors persist in the well after the enzyme-substratereaction has terminated. In a chemiluminescent ELISA, thesubstrate is the limiting reagent in the reaction; as it is exhausted,light production decreases and eventually ceases. A well-optimized procedure using the proper antibody dilutions willproduce a stable output of light, allowing consistent andsensitive detection of proteins. When the antibody is not dilutedsufficiently, too much enzyme is present and the substrate isused up quickly. A stable output of light will never be achieved.This is the single greatest cause of variability in chemilumi-nescent ELISAs. To avoid this problem, it is crucial to optimizethe amount of antibody used for detection. Antibody supplierstypically suggest a dilution range for using their antibody in anELISA. This dilution range is often appropriate for experimentsdetected with a relatively insensitive chromogenic substrate,but a much greater dilution is generally required for optimumperformance with a sensitive chemiluminescent substrate.

Table 8. Advantages of enhanced chemiluminescence.

Sensitive • Intense signal with low background• Requires less antigen and antibody

Fast • Rapid substrate processing• Signal generated within seconds

Nonhazardous • No health hazards• No waste disposal problems

Stable • Unlike radioisotopes, the shelf life is long• Store at 4°C

Large linear • Can detect a large range of protein concentrationsresponseQuantitative • Results can be scanned using an imaging device, such

as a CCD camera

H2O2 + +HRPLIGHT

*

@ 425 nm

NH

NH

O

ONH2

O

O

O

ONH2

O

O

O

ONH2

S/N

Ratio

Biotinylated-HRP, pg/well

ABTSOPDTMB

QuantaBlu™ Substrate

0 1,000 2,000 3,000 4,000

90

80

70

60

50

40

30

20

10

0

48



SuperSignal® ELISA Pico Chemiluminescent SubstrateExperience the same sensitivity in your luminometer that you’ve come to expect from all Pierce SuperSignal® Products.

SuperSignal® ELISA Pico Chemiluminescent Substrate is optimizedfor luminometer-based assays to generate an intense light signal.

Figure 19. Immediate light generation with SuperSignal® ELISA PicoChemiluminescent Substrate. 200 pg of biotinylated HRP or biotinylated APwere added to separate wells of Reacti-Bind™ NeutrAvidin™ Coated WhitePolystyrene Plates. The plates were then incubated for 30 minutes at roomtemperature (RT) on a plate shaker and then each well was washed threetimes with BupH™ Tris Buffered Saline. Working solutions of chemiluminescentsubstrates were prepared according to the manufacturers’ instructions. ForSuperSignal® ELISA Pico Chemiluminescent Substrate and anotherluminol-based system (Brand A), 100 µl of each substrate working solutionwas added to the appropriate plate well. For the dioxetane-based system,wells were washed with 1X Assay Buffer. Next, 100 µl of the dioxetaneworking solution was added to the appropriate plate well. All plates wereincubated on a plate shaker at RT for 1 minute. Plates were then read on aplate luminometer with a 0.2 second read time per well. Several readingswere taken over a 30-minute period.

Highlights:• Immediate light generation – intense signal is produced

immediately at room temperature or at 37°C• High signal:noise ratio – minimal background• Low picogram sensitivity – detect proteins in your ELISAs

down to the picogram levels• Room temperature storage – a consistent product with

ambient shipping and no need to store at 4°C• 8-hour working solution stability – consistent performance

of the working solution over an 8-hour period with only a10% decrease in activity at 24 hours

• Flexible – signal can be read in black or white opaque plates• Emits light at 425 nm

ReferencesBradley, K.A., et al. (2004). J. Biol. Chem. 278, 49342-49347. McKevitt, M., et al. (2003). Genome Res. 13, 1665-1674.


Product # Description Pkg. Size Price37070 SuperSignal® ELISA Pico 100 ml $195

Chemiluminescent Substrate*Includes: Luminol/Enhancer 50 ml

Stable Peroxide Buffer 50 ml*SuperSignal® Technology is protected by U.S. patent # 6,432,662.

RLU

Time (minutes)

Dioxetane-Based, Brand BLuminol-Based, Brand ASuperSignal® ELISA Pico

Chemiluminescent Substrate

0 10 20 30

700

600

500

400

300

200

100

0

Kinetic Analysis of 200 pg of Biotinylated HRP

HRP Substrates for ELISAExcellent sensitivity for use in luminometers.

Researchers looking for greater sensitivity in their ELISAs orany other solution-based assay are now able to use chemilu-minescent substrates for enzyme detection and quantification.These ELISAs can take place in either a test tube or a microplateand are quantified by measuring relative light units (RLU) in aluminometer. SuperSignal® ELISA Pico Chemiluminescent

Substrate was developed for researchers who need highsensitivity at an economical price. SuperSignal® ELISA FemtoMaximum Sensitivity Chemiluminescent Substrate uses animproved enhancer system that meets the needs of high-throughput screening and diagnostic customers. Both havetheir own unique features as listed in the table.

Table 9. Which substrate is right for you?

Working Solution StabilitySubstrate Detection Limits Kinetics at Room TemperatureSuperSignal® ELISA Femto Maximum • Quantitative to the femtogram level • Immediate light generation • 6-hour working solution stabilitySensitivity Substrate • 5- to 30-minute stability,

depending on HRP concentrationSuperSignal® ELISA Pico • Quantitative to the picogram level • Immediate light generation • 8-hour working solution stabilityChemiluminescent Substrate • 30-minute stable light output • Only 10% loss of activity after 24 hours


50


SuperSignal® ELISA FemtoMaximum SensitivitySubstrate utilizes theSuperSignal® Systemwith a new enhancerformulation for superiorprotein detection and low-end linearity in ELISAapplications.

SuperSignal® ELISA Femto Substrate’s rapid signal generationhas the added benefit of decreasing the substrate incubationperiod. SuperSignal® ELISA Femto Substrate generates detectablelight within 1 minute of addition to trace amounts of solubleHRP, saving up to 30 minutes per assay. This feature makesSuperSignal® ELISA Femto Substrate ideal for high-throughputscreening (HTS) applications in which as many as 100,000assays may be run daily on robotic equipment. As incubationperiods are often a limiting factor in the development of rapidautomated assays, SuperSignal® ELISA Femto Substrate is thelogical choice for automated HTS applications.

Highlights:• Immediate light generation – intense signal generated

immediately at both room temperature and 37°C• Improved low-end linearity – easy detection of low quantities

of proteins with high signal:noise ratios and low-end linearityof dose response curves

• High sensitivity – femtogram-level detection of targetproteins in an ELISA

• Reduction in assay time – high sensitivity allows for reductionin ELISA incubation steps

• Stability – storage for six months at room temperature or aminimum of 12 months at 4°C with a six-hour workingsolution stability

• Emits light at 425 nm

Incubation Time Required to Reach Maximum SignalSuperSignal® ELISA Femto 1 minute at room temperatureMaximum Sensitivity SubstrateSuperSignal® ELISA Pico 1 minute at room temperatureChemiluminescent SubstrateDioxetane-based Substrate System 30 minutes at room temperatureLuminol-based Substrate System 1 minute at room temperatureAcridan-based Substrate System 5 minutes at room temperatureTMB Colorimetric Substrate 15 minutes at room temperature

Figure 20. Femtogram detection of target protein and superior low-endlinearity. The dose response curve generated from an IL-2 ELISA illustratesthe exceptional low-end linearity achieved with SuperSignal® ELISA FemtoSubstrate and the incredible sensitivity attainable. The SuperSignal® ELISAFemto Substrate detected down to 168 fg of IL-2. The R2 value of the curvewas calculated to be 1.00 for signal generated at less than 1,600 fg of IL-2.

Figure 21. Signal intensity and kinetics comparison of chemiluminescentsubstrates. The dose response curve using SuperSignal® ELISA FemtoMaximum Sensitivity Substrate in an IL-2 ELISA was compared to curvesgenerated with a dioxetane-based substrate, another luminol-basedsubstrate, an acridan-based system and TMB. SuperSignal® ELISA FemtoMaximum Sensitivity Substrate demonstrated immediate light generationwith maximum peak intensity and high RLU values.

ReferencesBrandt, E.B., et al. (2003). J. Clin. Invest. 112, 1666-1677. Hanley, N.R.S. and Hensler, J.G. (2002). J. Pharmacol. Exp. Ther. 300, 468-477.Masri, H.P. and Cornellissen, C.N. (2002). Infect. Immun. 70, 732-740.Su, S.V., et al. (2004). J. Biol. Chem. In press.


Product # Description Pkg. Size Price37075 SuperSignal® ELISA Femto 100 ml $205

Maximum Sensitivity Substrate*Includes: Luminol/Enhancer 50 ml

Stable Peroxide Solution 50 ml*SuperSignal® Technology is protected by U.S. patent # 6,432,662.

Net R

LU

pg IL-20 5 10 15 2520

1,500

1,200

900

600

300

0

SuperSignal® ELISA Femto Substrate SuperSignal® ELISA Pico Substrate

Dioxetane-based SubstrateLuminol-based SubstrateAcridan-based Substrate

Net R

LU

fg IL-20 500 1,000 1,500

120

100

80

60

40

20

0

SuperSignal® ELISA Femto Maximum Sensitivity SubstrateThe most powerful substrate for high-throughput screening/diagnostic applications with high sensitivity and superior low-end linearity.

51

QuantaBlu™ Fluorogenic Peroxidase SubstratesThe ideal fluorescent substrates for use with peroxidase enzymes.

A variety of substrates areavailable for detectingperoxidase activity inELISA-based assays.Colorimetric substrates(e.g., TMB, OPD andABTS) have been usedwidely for years. Each ofthese substrates variesgreatly with respect to its

performance characteristics such as detection sensitivity,working range and attainable signal:noise ratios. Substrateflexibility is also a key issue that affects an assay. Stability,development time requirements and the capacity to performstopped and/or kinetic assays vary significantly among thesesubstrates. Ideally, a substrate is stable, is very sensitive, hashigh attainable signal:noise ratios, possesses a broad dynamicrange, and allows the user to perform stopped, nonstoppedand kinetic assays. QuantaBlu™ Fluorogenic Peroxidase Substratemeets all the requirements of an ideal substrate for use inperoxidase detection.

QuantaBlu™ Substrate generates a blue fluorescent productupon reaction with peroxidase. The fluorescent product doesnot photobleach. Fluorometric-based detection overcomes thelimitations of colorimetric substrate detection, which does notallow for quantitation of greater than four optical density units.QuantaBlu™ Substrate allows for stopped, nonstopped and kineticassays to be performed. Incubation times with QuantaBlu™

Substrate for stopped and nonstopped assays may be variedbetween 1 to 90 minutes at either room temperature or 37°C.QuantaBlu™ Substrate exhibits a flat baseline in assays, whichfacilitates low-level detection sensitivity and allows for highsignal:noise ratios.

QuantaBlu™ Fluorogenic Peroxidase Substrate provides the abilityto rapidly detect peroxidase at very low concentrations in shortperiods of time. Figure 23 illustrates the detection of peroxidasefrom 0-10 pg per well from 1.5 to 6.5 minutes of substrateincubation time. At cycle 1 (1.5 minute incubation) as little as2.5 pg of peroxidase could be detected, while at cycle 6 (6.5minute incubation) 0.625 pg of peroxidase could be detected.

Highlights:• More sensitive than TMB, OPD or ABTS substrates• Flexible stopped, nonstopped or kinetic assays possible• Large dynamic range (4 log peroxidase concentration range)• Excellent stability – working solution is stable for 24 hours• Large Stokes’ shift; excitation/emission maxima of 325/420;

range of 315-345/370-460• Does not photobleach

Figure 22. Comparison of QuantaBlu™ Substrate to other substrates.QuantaBlu™ Substrate and the colorimetric substrates were incubated for 30minutes at RT, followed by addition of a stop solution. QuantaBlu™

Substrate gave the greatest signal:noise (S/N) ratios and exhibited thelowest detection limit.

Figure 23. Rapid and sensitive detection with QuantaBlu™ FluorogenicPeroxidase Substrate. Detection of bound biotinylated peroxidase at 0-10pg/well using QuantaBlu™ Substrate. QuantaBlu™ Fluorogenic PeroxidaseSubstrate was read in a nonstopped mode using a 1-minute instrumentcycle time between reads.

ReferencesAtamna, H., et al. (2000). Proc. Natl. Acad. Sci. 97, 686-691. Ayala, P., et al. (2002). Infect. Immun. 70, 5965-5971. Jefcoat, A.M., et al. (2001). Am J Physiol Lung Cell Mol Physiol. 281, L704-712. Savage, M.D., et al. (1998). Previews 2(1), 6-9.Savage, M.D., et al. (1998). Previews 2(2), 18-19.


Product # Description Pkg. Size Price15169 QuantaBlu™ Fluorogenic Kit $169

Peroxidase Substrate*Includes: QuantaBlu™ Substrate 250 ml

QuantaBlu™ Stable Peroxide Solution 30 mlQuantaBlu™ Stop Solution 275 ml

15162 QuantaBlu™ NS/K Substrate Kit $140(for nonstopped and kinetic assays)Includes: QuantaBlu™ Substrate 250 ml

QuantaBlu™ Stable Peroxide Solution 30 ml* QuantaBlu™ Fluorogenic Peroxidase Assay Technology is protected by

U.S. patent # 6,040,150.

S/N

Ratio


4 minutes5 minutes6 minutes

1 minutes2 minutes3 minutes

0 2 4 6 8 10

4.0

3.5

3.0

2.5

2.0

1.5

1.0

S/N

Ratio


ABTSOPDTMB

QuantaBlu™ Substrate

0 1,000 2,000 3,000 4,000

90

80

70

60

50

40

30

20

10

0

OPD

OPD yields a water-soluble yellow-orange product when reactedwith peroxidase with an absorbance maximum of 492. OPDcan easily be dissolved in a substrate buffer such as StablePeroxide Substrate Buffer (Product # 34062).


Product # Description Pkg. Size Price34005 OPD 25 g powder $ 4534006 OPD Tablets 50 tablets (5 mg/tablet) $104

ABTS

ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is a water-soluble HRP substrate that yieldsa green end product upon reaction with peroxidase. The greenproduct has two major absorbance peaks, 405 nm and 650 nm.ABTS is less sensitive than OPD and TMB in ELISA applications.It is less readily oxidized, and its color development is slower(approximately 20 minutes). This may be advantageous ifunacceptable background results from the use of the OPD orTMB substrates due to higher sensitivities.

Highlights:• One component• Ready-to-use• Excellent choice when maximum sensitivities are not required• Slow reaction that can easily be followed with a kinetic reader

ReferencesPaing, M.M., et al. (2002). J. Biol. Chem. 277, 1292-1300. Sau-Ching Wu, S.-C., et al. (2002). Appl. Envir. Microbiol. 68, 3261-3269.


Product # Description Pkg. Size Price34026 ABTS 50 tablets (10 mg/tablet) $ 8137615 1-Step™ ABTS 250 ml (Ready-to-use) $ 70

52

CHOOSING A SUBSTRATEColorimetric Substrates for HRP

KineticMeasurement Absorbance

Description Highlights Sensitivity Possible Maximum Color Product #1-Step™ ABTS • One component Low Yes 405 nm Green 37615

• Ready to use• Excellent choice when maximum sensitivities are not required• Slow reaction that can be easily followed with a kinetic reader

ABTS Tablets Low Yes 405 nm Green 34026OPD Tablets • OPD can be easily dissolved in a substrate buffer such as High No 492 nm Yellow- 34006

Stable Peroxide Substrate Buffer (10X) (Product # 34062) orangeOPD Powder High No 492 nm Yellow- 34005

orangeTMB Substrate Kit • Highest sensitivity Very High No 450 nm Yellow 34021

• Easy to use• Results in seconds• No DMF or DMSO in reagent

1-Step™ Ultra • One component Very High No 450 nm Yellow 34028TMB-ELISA • Ready to use

• Highest sensitivity• No DMF or DMSO in the reagent

1-Step™ Turbo TMB • One component High No 450 nm Yellow 34022• Ready to use• No DMF or DMSO in the reagent

1-Step™ Slow TMB • One component Medium Yes 450 nm Yellow 34024• Ready to use• No DMF or DMSO in the reagent


TMB

TMB is a chromogen thatyields a blue color (measur-able at 370 nm or 652 nm)when oxidized with hydrogenperoxide (catalyzed by HRP).The color then changes toyellow (measured at 450 nm)upon addition of sulfuric orphosphoric acid to stop the

reaction. TMB is very sensitive and more quickly oxidized thanother HRP substrates, resulting in faster color development.

The 1-Step™ TMB Substrates are one-component substrates thatrequire no preparation before use. Unlike other commerciallyavailable substrates, these products contain no DMF or DMSO.There are three formulations that differ primarily in their sensi-tivities. 1-Step™ Slow TMB is intermediate in sensitivity – idealfor kinetic readings. The sensitivity of the 1-Step™ Turbo TMBcompares to that of OPD used at approximately 1 mg/ml. 1-Step™

Ultra TMB-ELISA produces the highest signal:noise ratio andsensitivity in the picogram range.

Highlights:• Ready-to-use single component• No hydrogen peroxide required• No filtering required• Noncarcinogenic• Various sensitivities to suit any assay

Figure 24. Comparison of sensitivities of Turbo TMB, Slow TMB and ABTS.

Figure 25. 1-Step™ Ultra TMB-ELISA provides more signal than otherTMB substrates.

Figure 26. 1-Step™ Ultra TMB-ELISA produces higher signal:noise (S/N)ratios than other TMB substrates.

ReferencesHong, P.W., et al. (2002). J. Virol. 76, 12855-12865.Murphy, M.B., et al. (2003). Nucleic Acids Res. 31, e110. Su, S.V., et al. (2004). J. Biol. Chem. In press.Tek, V. and Zolkiewski, M. (2002). Protein Sci. 11, 1192-1198. Thomas, P.E., et al. (1976). Anal. Biochem. 75, 168-176.Weimer, B.C., et al, (2001). Appl. Envir. Microbiol. 67, 1300-1307. Wu, S.-C. and Wong, S.-L. (2002). Appl. Envir. Microbiol. 68, 1102-1108.


Product # Description Pkg. Size Price34024 1-Step™ Slow TMB 250 ml $ 6634022 1-Step™ Turbo TMB 250 ml $ 9134028 1-Step™ Ultra TMB-ELISA 250 ml $10134021 TMB Substrate Kit Kit $102

Includes: Peroxidase Substrate (TMB) 200 mlPeroxide Solution (H202) 200 ml

S/N

Ratio

TMB Sources Used

9.0

7.5

6.0

4.5

3.0

1.5

0

Pierce

1-Step

™

Ultra TM

B – EL

ISA

Compe

titor B

Compe

titor D

Compe

titor N

Compe

titor K

Compe

titor I

Compe

titor M

Abso

rban

ce a

t 450

nm

Concentration of Human IFN Gamma (pg/ml)50 100 150 200 250 300 350 400 450 500

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Pierce 1-Step™ Ultra TMB - ELISACompetitor B

Competitor DCompetitor ICompetitor K

Competitor MCompetitor N

0

ABTSSlow TMBTurbo TMB

1.5

1.0

0.5

0.0

Abso

rban

ce a

t 450

nm

(TM

B)Ab

sorb

ance

at 4

10 n

m (A

BTS)

Dilution of HRP Conjugate(from 1 mg/ml stock)

1.56 x 10-5 6.25 x 10-5 1.25 x 10-4 2.5 x 10-4

3.13 x 10-5

54


PNPPDetection of alkaline phosphatase label in ELISA applications.

PNPP (p -Nitrophenyl Phosphate, Disodium Salt) is a widelyused substrate for detecting alkaline phosphatase in ELISAapplications.1 When alkaline phosphatase and PNPP arereacted, a yellow water-soluble reaction product is formed.This product absorbs light at 405 nm. Pierce offers PNPP infour formats. PNPP is available either as a crystalline powderor 5 mg tablets. Also available is the Phosphatase SubstrateKit, which contains PNPP tablets and a Diethanolamine Bufferto yield more than one liter of substrate. The DiethanolamineSubstrate Buffer (Product # 34064) is also provided individuallyas a 5X concentrate. The Pierce formulation has an optimaland consistent pH and it is stable even at a 1X concentration.

Until recently, the method for preparing PNPP solutions requireddissolving the powder or tablets in buffer and then diluting thesolution to the desired concentration. Substrate instability madeit necessary to prepare it on a daily basis. Pierce 1-Step™ PNPPcircumvents this time-consuming and variable-introducingstep by providing a single-component PNPP substrate. Thissubstrate is stable for 12 months at 2-8°C and can be stoppedwith conventional methods.

Reference1. Snyder, S.L., et al. (1972). Biochim. Biophys. Acta 258, 178-187.Bosque, P.J., et al. (2002) Proc. Natl. Acad. Sci. 99, 3812-3817. Jan, J.-T., et al. (2000) J. Virol. 74, 8680-8691.


Product # Description Pkg. Size Price34045 PNPP 25 g powder $10034047 PNPP Tablets 105 tablets $102

(5 mg/tablet)37620 Phosphatase Substrate Kit Kit $135

Sufficient reagents for 1.05 liters of substrate.Includes: Diethanolamine Buffer 225 ml

PNPP Tablets 105 tablets(5 mg/tablet)

37621 1-Step™ PNPP 100 ml $ 77

Alkaline Phosphatase Substrates

Alkaline phosphatase, a 140 kDa protein that is generallyisolated from calf intestine, catalyzes the hydrolysis of phosphategroups from a substrate molecule, resulting in a colored orfluorescent product or the release of light as a byproduct. APhas optimal enzymatic activity at a basic pH (pH 8-10) and canbe inhibited by cyanides, arsenate, inorganic phosphate anddivalent cation chelators, such as EDTA. As a label for ELISA,AP offers a distinct advantage over other enzymes. Since itsreaction rate remains linear, detection sensitivity can be improvedby simply allowing a reaction to proceed for a longer time period.Due to the large size of AP, it is very sensitive to freeze/thawand should be aliquotted and stored at 4°C.

The most common ELISA substrate for alkaline phosphataseis the chromogen p -nitrophenyl phosphate (PNPP), which isavailable in several formats. PNPP yields a yellow reactionproduct that is water-soluble and absorbs light at 405 nm.The 1-Step™ PNPP Substrate (Product # 37621) offers theconvenience of a ready-to-use reagent with similar sensitivityto the two-component kit. The Phosphatase Substrate Kit(Product # 37620) includes PNPP tablets and diethanolaminebuffer (5X). PNPP is also available as 25 g of powder (Product# 34045) or in tablet form (Product # 34047). DiethanolamineSubstrate Buffer (Product # 34064) is a convenient, ready-to-useformulation sold as a 5X concentrate with an optimal andconsistent pH. Soluble ELISA substrates for AP are summarizedin Table 7, on page 46.

Kinetic Measurement Absorbance Description Features/Benefits Sensitivity Possible Maximum Color Product #1-Step™ PNPP • One component High Yes 405 nm Yellow 37621

• Ready to use• Stable for 12 months• The easiest-to-use PNPP substrate available

Phosphatase • Ideal for ELISA High Yes 405 nm Yellow 37620Substrate Kit • Easy to use

• Minimal assay-to-assay variability• Low background

PNPP Tablets High Yes 405 nm Yellow 34047PNPP Powder High Yes 405 nm Yellow 34045

Substrate BuffersSubstrate buffers for alkaline phosphatase and horseradish peroxidase.

Buffered stable peroxide is used by researchers who prefer tomake their own HRP-ELISA substrates. This is done easily byadding a chromogen of choice to the buffer. The Stable PeroxideSubstrate Buffer available from Pierce has a long shelf life(12 months after receipt), and is provided as a 10X concentrate.A total volume of 1,000 ml can be made from one 100 ml bottleof concentrate. If you use 10 ml of substrate per plate, you canmake substrate for 100 plates from only one bottle of StablePeroxide Substrate Buffer. Diethanolamine Substrate Bufferis used with soluble alkaline phosphatase substrates such asPNPP. Our formulation is convenient, ready-to-use and reducesthe possibility of assay-to-assay variability. DiethanolamineSubstrate Buffer is sold as 5X concentrate with an optimal,consistent pH. It is stable even at a 1X concentration.

Highlights:• Ready-to-use• Minimize assay-to-assay variability

ReferencesLouis, H., et al. (2000) J. Histochem. Cytochem. 48, 499-508. Neisewander, J.L., et al. (2000) J. Neurosci. 20, 798-805. Pifer, J., et al. (2002) J. Immunol. 169, 1372-1378.


Product # Description Pkg. Size Price34062 Stable Peroxide Substrate Buffer (10X) 100 ml $ 4034064 Diethanolamine Substrate Buffer (5X) 225 ml $ 47

ONPG Colorimetric β-Galactosidase Soluble SubstrateFor detection of β-Galactosidase label in ELISA applications.

β-Galactosidase Soluble Colorimetric SubstrateKineticMeasurement Absorbance

Description Highlights Sensitivity Possible Maximum Color Product #ONPG • Forms a completely soluble product when reacted with β-Galactosidase High Yes 420 nm Yellow 34055

• High extinction coefficient at 405 nm• Yields a yellow product easily detectable in the visual range

When using β-Galactosidase as the label for proteins in ELISAstudies, a wide variety of substrates are available, includingo -nitrophenyl-β-D-galactopyranoside (ONPG), naphthol-AS-BI-β-D-galactopyranoside (Nap-Gal) and 4-Methyl-umbelliferyl-β-D-galactopyranoside (Mum-Gal). However, it is important tochoose a substrate with adequate solubility, that uses readilyavailable equipment and that gives a significant reading overthe background. ONPG is a superior β-Galactosidase substrateoption.1 The product formed is completely soluble and has ahigh extinction coefficient at 405 nm. The substrate yields ayellow product that is easily detectable in the visual range afterstopping the reaction with 1 M sodium carbonate.

Reference1. Craven, G.R., et al. (1965). J. Biol. Chem. 240, 2468-2477.


Product # Description Pkg. Size Price34055 ONPG 5 g powder $ 63


Easy-Titer® ELIFA System

The Easy-Titer® ELIFA System offers faster ELISA results byoverriding limiting diffusion, which is the rate limiting step inELISA.1,2 Limiting diffusion is a phenomenon in which solublereactants are depleted near the reaction surface, and the rateof reaction is limited by diffusion of new reactants to the site.3The enzyme-linked immunoflow assay (ELIFA) system actuallyreduces one-hour incubations to just a five-minute filtration step.A vacuum pulls substrates through a transfer membrane thathas complementary ligands or enzymes bound to it. Cannulasprecisely and quantitatively transfer unbound substrate to thecollection chamber and transfer colored product into corre-sponding microwells (Figure 27) for analysis in an automatedmicroplate reader. Assay times required for the Easy-Titer® ELIFASystem and a traditional ELISA are compared in Table 8.

Highlights:• Allows complete ELISA-type assays to be performed in just

25 minutes• Offers greater sensitivity because transfer membranes bind

more protein than plastic surfaces• 96-well arrangement is compatible with 96-well microplates

and multi-channel pipettes• Most wash steps are eliminated, further reducing assay time• No cross-contamination between samples because of tight seals• Results are easily quantitated

Figure 27. Easy-Titer® ELIFA System. Colored product is transferred tomicroplate wells for an ELISA or precipitated on the membrane for a dot blot.

Table 8. Comparison of assay times for Easy-Titer® ELIFA System and standard ELISA.

Faster Than ELISA Easy-Titer® System ELISAAdsorb antibody 5 minutes 1 hourBlock empty sites 5 minutes 30-60 minutesBind antigen 5 minutes 1 hourAntibody-enzyme 5 minutes 1 hourProduce color 5 minutes 10-30 minutesTotal Assay Time 25 minutes 3.6-4.5 hours

References1. Engvall, E. and Perlmann, P.O. (1971). Immunochemistry 8, 871-875.2. Palomäki, P. (1991). J. Immunol. Method 145, 55-63.3. Rao, P.N. and Taraporewala, I.B. (1992). Steroids 57, 154-161.4. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170.5. Weller, T.H. and Coons, A.H. (1954). Proc. Soc. Exp. Biol. (New York) 86, 789-794.6. Schwab, C. and Bosshard, H.R. (1992). J. Immunol. Method 147, 125-134.7. Stenberg, M. and Nygren, H. (1988). J. Immunol. Method 113, 3-15.8. Valkirs, G.E. and Barton, R. (1985). Clin. Chem. 31, 1427-1431.9. Ijsselmuiden, O.E., et al. (1989). J. Immunol. Method 119, 35-43.

Collection chamber

Transfer cannula

GasketsMembrane

Sample application plate

Sample well

Microplate

56

ELIFASYSTEM


Easy-Titer® ELIFA SystemReduces each step to a five-minute filtration!

Highlights:• Effective ELISA device

allows immunoassaysto be completed in just25 minutes (each stepis reduced to a 5-minutefiltration)

• Ideal for ELISA-typeimmunoassays, dot blotsand nucleic acid blots

• Binding kinetics of any ELISA step performed using Easy-Titer®

System overrides limiting diffusion• Greater sensitivity because transfer membranes bind more

protein than plastic surfaces• 96-well arrangement is compatible with 96-well microplates

and multichannel pipettes• Most wash steps are eliminated, further reducing assay time• Cross-talk between samples is eliminated due to the use of

tight seals• Results are quantitated easily by simply drawing color solution

into a microplate and reading using a standard ELISA reader• Use insoluble or precipitating substrates to create dot blot results

Figure 28. A checkerboard titration is a single experiment in which theconcentration of two components is varied in a way to visualize the bestsignal:noise ratio conditions.

References1. Paffard, S.M., et al. (1996). J. Immunol. Method 192, 133-136.Liu, Z.-H., et al. (2001). Proc. Natl. Acad. Sci. 98, 7289-7294. Menalled, L.B., et al. (2002). J. Neurosci. 22, 8266-8276.


Product # Description Pkg. Size Price77000 Easy-Titer® ELIFA System* $1,011

Includes: Sample application plateTop silicone gasketMembrane support plate with bottom silicone gasket and 96 cannulas

Vacuum chamber with o-ringThumbscrews 4One-way valves 4Valve/tubing adapters 4Tubing 4 ftExtra cannulas 10

77015 Biodyne® A Nylon Membranes 25/pkg. $ 26577016 Biodyne® B Nylon Membranes 25/pkg. $ 19077070 Easy-Titer® Replacement Cannulas 100/pkg. $ 5777071 Easy-Titer® Replacement 4/pkg. $ 104

96-Well Clear Silicone Gaskets* The Easy-Titer ® ELIFA System is protected by U.S. patent # 5,219,528.

1:1 1:2 1:4 1:8 1:16 1:32 1:64

1:1

1:2

1:4

1:8

1:16

1:32

1:64

1 2 2 3 4 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Primary Antibody Dilution

Labe

led

Seco

ndar

y An

tibod

y Di

lutio

n

57ELIFASYSTEM

58

BULK AND CUSTOMOFFERINGS

Coated Microplates (96-well or 384-well)

Partner with Pierce!Pierce can also help you commercialize your ELISA kit. We have the expertise, manufacturing capability and ISO 9001 certificationto help you launch a new dimension to your business. Our scientific staff will work with your research team to implement world-classproject management and meet your deadlines for a new product launch. Partner with Pierce to reach the next level of commercialsuccess for your company.

* Minimum quantities apply based on largest catalog package size available. Call for more information.

Need more product than our catalog package size offers?

Contact the Bulk & Custom Products Department at Pierce or your local distributor torequest a price quote that will satisfy your large-scale needs. All of our popular ELISAproducts are available in bulk quantities* and in special package sizes. Delivery can bearranged to meet your schedule. Pierce can bring all of these critical componentstogether to help you develop your own ELISAs on a grand scale:

Blocking Buffers• Serum-based Blockers • Non-serum Blockers• Milk-based Buffers

Immunoassay Buffers• Sample Preparation Buffers • Wash Buffers• Assay Reagent Diluents • Stop Solutions

Soluble Substrates• Colorimetric • Chemiluminescent• Fluorescent

Enzyme Conjugate Stabilizer

Immunoassay Conjugates• Antibody-enzyme Conjugates • NeutrAvidin™-enzyme Conjugates• Biotinylated Antibody Conjugates • Protein A, G, L or A/G-enzyme Conjugates• Streptavidin-enzyme Conjugates • Nickel-chelate Enzyme Conjugate

800-874-3723 • 815-968-0747 • www.piercenet.com 59BULKAND CUSTOMOFFERINGS

ChoiceCoat® Custom Microplate Packaging OptionsPierce can provide the appropriate packaging for your custommicroplates, based upon your usage. Coated microplates canbe packaged for large-screening applications (i.e., packaged in25 plates ready for stacking) or for inclusion in a final kit forresale (i.e., single-pouch packages). Stability testing forcustom microplates can be determined if long-term storage iscritical for your application. Scheduled shipments can beestablished easily to make the microplates available when youneed them most.

Minimum Quantities for ChoiceCoat® Custom Microplate-coating Service Get started creating your own custom microplate with as fewas 500 (96-well) microplates or 200 (384-well) microplates.Pierce will provide five coated microplates at no charge fortesting purposes before we ship any lot of ChoiceCoat®

Custom Coated Plates to you. Pierce will work closely withyou to develop the appropriate quality assurance tests so thatwe provide the most consistent product to meet your needs.

In the United States, contact the Pierce Bulk & Custom SalesDepartment at 1-800-874-3723 or 815-968-0747. Outsidethe United States, contact your local Perbio Science office orPierce distributor for a FREE ChoiceCoat® Consultation.

Partner with Pierce to discover the best choices!

The choice is up to you!

The ChoiceCoat® Custom Microplate-coating Service from PiercePierce has been coating plates for more than 10 years. Putthis experience and expertise to work for you!

Pierce can process up to 7,500 microplates per day and hasdeveloped novel chemistries and coatings for a number ofapplications. Following are just a few of the applications inwhich Pierce custom-coated microplates have been effectivein generating valuable results.

• Competitive LigandBinding Assays

• Oligonucleotide Binding Assays • Protease Assays• Kinase and Phosphatase Assays• Sandwich ELISAs• Pull-down Assays and Immunoprecipitation

ChoiceCoat® Microplate Coating Options Available

Microplate Type/Manufacturer Coat Protein/Ligand Blocking BufferBlack (96- or 384-well) Antibodies SuperBlock® BlockerWhite (96- or 384-well) Bacterial Fusion Proteins StartingBlock™ BlockerClear (96- or 384-well) Oligonucleotides or Peptides Purified CaseinClear bottom, Black Biological Polymers BSAClear bottom, White Metal Chelates SerumYour Microplate Your Ligand Your BlockerNote: Covalent chemistries also an option!

800-874-3723 • 815-968-0747 • www.piercenet.com60

RECOMMENDED READING

Antibodies: A Laboratory ManualMore than 700 pages of valuable information about antibodies and antibody production.

This valuable manual contains 726pages of information about the theory,production and use of monoclonal andpolyclonal antibodies. Oversized printand colorful graphics make it easy toread and understand.


Product # Description Price15051 Antibodies: A Laboratory Manual $132

Harlow, E. and Lane, D.,Published by Cold Spring Harbor Laboratory, 1988,726 pages, Softcover

ELISA Theory and PracticeBasic principles and techniques.

The ELISA Guidebook presents theprinciples of the assay technique calledenzyme linked immunosorbent assay.The book gives detailed descriptions ofthe basic ELISA principles and techniquesthat will aid both novice and experiencedresearchers in developing their ownrapid and accurate assays.

This 421-page book features thefollowing range of topics:

• Direct, indirect and competitive ELISA• Capture ELISA on solid-phase• Basic immunology• Stages in ELISA• Theoretical considerations• Practical exercises• Immunochemical techniques


Product # Description Price15056 The ELISA Guidebook $100

Crowther, J.R., Published by Humana Press, Inc., 2001,421 pages, Softcover

Protein MethodsTreats practical protein-related issues that most other books do not address.

This expanded second edition providesstep-by-step explanations of basicmethods for extracting, handling,storing, measuring, solubilizing andconcentrating proteins.


Product # Description Price20001 Protein Methods $ 78

Bollag, D.M., et al., Published by Wiley-Liss, Inc., 1996,400 pages, Comb-bound

RECOMMENDEDREADING 61

Protein Assay Technical Handbook

The 38-page Protein Assay TechnicalHandbook is a helpful guide to choosingthe best protein assay and standard for asample. It contains abundant informationon the most widely used protein assaymethods (BCA™, Lowry and BradfordAssays), including the principle behindeach protein assay and its uniqueadvantages and disadvantages. The

handbook also includes methods for detection specific proteintypes such as histidine-tagged proteins, antibodies and proteases.


Product # Description Price1601004 Protein Assay Technical Handbook FREE

Western Blotting Handbook and Troubleshooting Guide

This 52-page handbook containsfull-length and abbreviated Westernblotting protocols, tips on antibody andblocking buffer optimization, a chemilu-minescent substrate selection guide, aprocedure for stripping and reprobingWestern blots, tips on clearer filmdevelopment, and other information thatwill be useful to experts and first-timers

alike. Learn more about four new Western blotting products thatcan improve your blot’s signal:noise ratio: Restore™ Western BlotStripping Buffer, Qentix™ Western Blot Signal Enhancer, Erase-It®

Background Eliminator and StartingBlock™ Blocking Buffers.


Product # Description Price1600990 Western Blotting Brochure FREE

Antibody Technical Handbook

This new 68-page handbook helps youchoose the best methods to produce,purify, fragment and label antibodies.Topics include basic immunology,carrier proteins, adjuvants, antibodypurification methods, antibody frag-mentation with proteases, and labelingantibodies with a variety of tags (e.g.,biotin, fluorophores, enzymes, iodine)for purification or detection.


Product # Description Price1601092 Antibody Technical Handbook FREE

Sorry, books are nonreturnable.

G r a s p t h e P r o t e o m e ™

Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316Customer Assistance E-mail: [email protected]

Outside the United States, visit our web site or call 815-968-0747 to locate your local Perbio Science branch office (below) or distributor

© Pierce Biotechnology, Inc., 2004. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only.Prices listed herein are accurate at time of printing. CyDye™, Cy3™ and Cy5™ are registered trademarks of Amersham Biosciences. Tween® and Brij® are registered trademarks of ICI Americas.

Kathon® and Triton® are registered trademarks of Rohm & Hass Co. Biodyne® is a trademark of Pall Corporation. Texas Red® is a trademark of Molecular Probes, Inc.

Belgium & Dist.:Tel +32 (0)53 83 44 04

[email protected]

China:Tel: (8610)8048 [email protected]

France:Tel 0800 50 82 15

[email protected]

Germany: Tel 0228 9125650

[email protected]

Hong Kong:Tel : 852 2753 [email protected]

The Netherlands: Tel 076 50 31 880

[email protected]

United Kingdom: Tel 0800 252185

[email protected]

Switzerland: Tel 0800 56 31 40

[email protected]

SuperSignal® ELISA Substrates• Strong, sensitive signal – clear detection

in the femtogram range• Low noise – no auto-generated back-

ground like many fluorescent systems• Immediate signal generation – no

waiting for detection• Stable light output – no drop in signal

for up to 30 minutes

StartingBlock™ Buffers• Limits background when others don’t –

guaranteed serum protein- and biotin-free formulations

• Does not cross-react with rabbit anti-bodies – a common problem withother blockers

• “No-wait” blocking – just pipette and remove

• Convenient – buffers are available withor without Tween®-20 Detergent added

Reacti-Bind™ Coated Plates• Pre-coated plates – for binding, anti-

bodies, fusion proteins and biotin orfor covalently binding targets

• Selective – plates bind only the targetligand, not unwanted, noise-producingmolecules

• Convenient normal- and high-bindingversions are available

www.piercenet.com

Turn down the noise!Three ways to get the highest signal:noise ratio possible.

Pump up the volume!

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