OverviewOften it is useful to study or make use of the activity

of one portion of an immunoglobulin without interference

from other portions of the molecule. It is possible to selectively cleave

the immunoglobulin molecule into fragments that have discrete characteristics.

Antibody fragmentation is accomplished using proteases that digest or cleave certain

portions of the immunoglobulin protein structure. Although fragmentation of all

immunoglobulin classes is possible, only procedures for fragmentation of mouse, rabbit and

human IgG and IgM have been well-characterized.

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Antibody Fragmentation

The two groups of antibody fragments of primary interest areantigen-binding fragments such as Fab and nonantigen-binding,class-defining fragments such as Fc. More than one type ofantigen-binding fragment is possible, but each contains at leastthe variable regions of both heavy and light immunoglobulin chains(VH and VL, respectively) held together (usually by disulfide bridges)so as to preserve the antibody-binding site. Fc fragments consistof the heavy chain constant region (Fc region) of an immunoglobulinand mediate cellular effector functions.

Antibody fragmentation is somewhat laborious, requires optimiza-tion of enzyme-mediated digestion of the protein and necessitatesan ample supply (e.g., 10 mg) of antibody to make it reasonablyefficient. For these reasons, fragmentation is usually performedonly when the antibody of interest is available in large quantityand the particular application demands it.

Advantages of AntibodyFragments Antibody fragments offer several advantages over intact antibodyas reagents in an immunochemical technique:• Using antigen-binding regions that have been separated from

the Fc region reduces nonspecific binding that results from Fcinteractions (many cells have receptors for binding to the Fcportion of antibodies).

• Small antigen-binding fragments generally provide highersensitivity in antigen detection for solid-phase applications as aresult of reduced steric hindrance from large protein epitopes.

• Because they are smaller, antibody fragments more readilypenetrate tissue sections, resulting in improved staining forimmunohistochemical applications.

• Antibody fragments are the best choice for antigen-antibodybinding studies in the absence of Fc-associated effectorfunctions (e.g., complement fixation, cell membrane receptorinteraction).

• Antibody fragments offer a simple system by which to studythe structural basis for immune recognition using X-ray crystallography or nuclear magnetic resonance.

• Antibody fragments have lower immunogenicity than intactantibody.

Types of AntibodyFragmentsF(ab′)2, Fab, Fab′ and Fv are antigen-binding fragments that canbe generated from the variable region of IgG and IgM. Theseantigen-binding fragments vary in size (MW), valency and Fccontent. Fc fragments are generated entirely from the heavy chainconstant region of an immunoglobulin. The structures of theseantibody fragments are illustrated in schematic form in Figure 19and summarized below. In addition, several unique fragmentstructures can be generated from pentameric IgMs, includingan “IgG”-type fragment, an inverted “IgG”-type fragment and apentameric Fc fragment. IgM fragmentation is discussed in detailon pages 48-49.

F(ab′)2

F(ab′)2 (110,000 dalton IgG fragment, 150,000 dalton IgM fragment)fragments contain two antigen-binding regions joined at the hingethrough disulfides. This fragment is void of most, but not all, ofthe Fc region.

Fab′Fab′ (55,000 dalton IgG, 75,000 dalton IgM) fragments can beformed by the reduction of F(ab′)2 fragments. The Fab′ fragmentcontains a free sulfhydryl group that may be alkylated or utilizedin conjugation with an enzyme, toxin or other protein of interest.Fab′ is derived from F(ab′)2; therefore, it may contain a smallportion of Fc.

FabFab (50,000 daltons) is a monovalent fragment that is producedfrom IgG and IgM, consisting of the VH, CH1 and VL, CL regions,linked by an intrachain disulfide bond.800-874-3723 • 815-968-0747 • www.piercenet.com

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AntibodyFragmentation

FvFv (25,000 daltons) is the smallest fragment produced from IgGand IgM that contains a complete antigen-binding site. Fv fragmentshave the same binding properties and similar three-dimensionalbinding characteristics as Fab. The VH and VL chains of the Fvfragments are held together by noncovalent interactions. Thesechains tend to dissociate upon dilution, so methods have beendeveloped to cross-link the chains through glutaraldehyde,intermolecular disulfides or a peptide linker.

“r IgG”“r IgG” (80,000 daltons) is a reduced form of IgG composed of onecomplete light chain and one complete heavy chain. It is essentiallyone-half of an intact IgG molecule and it contains a single antigen-binding site. “r IgG” fragments are formed by the selectivereduction of disulfide bonds in the hinge region of an antibody.

FcFc (50,000 daltons) fragments contain the CH2 and CH3 region andpart of the hinge region held together by one or more disulfidesand noncovalent interactions (Figure 19). Fc and Fc5µ fragmentsare produced from fragmentation of IgG and IgM, respectively.The term Fc is derived from the ability of these antibody fragmentsto crystallize. Fc fragments are generated entirely from the heavy-chain constant region of an immunoglobulin. The Fc fragmentcannot bind antigen, but it is responsible for the effector functionsof antibodies, such as complement fixation.

F(ab′)2, Fab′, Fab and Fv fragments produced from IgM functionin much the same way as F(ab′)2, Fab′, Fab and Fv fragments fromIgG. However, compared to those in IgG, individual antigen-bindingsites in IgM generally have lower binding affinities, which arecompensated in the complete IgM by its pentameric form. Theincreased binding valency of F(ab′)2 may make it preferable toFv and Fab fragments.

F(ab′)2 fragments are divalent, and they may be a superior alter-native to Fab fragments for antibodies with low affinity. The F(ab′)2

fragments have higher avidity than the Fab and Fab′ fragments.F(ab′)2 fragments can precipitate antigen. Fab and Fab′ areunivalent molecules that cannot precipitate antigen. Fab andFab′ fragments have a decreased binding strength, and normallystable antigen-antibody complexes may dissociate duringwashes in certain applications.

Fragmentationof IgGThe hinge region of an immunoglobulinmonomer (IgG) is readily accessible to proteo-lytic attack by enzymes. Cleavage at this pointproduces F(ab′)2 or Fab fragments and the Fcfragment. The Fc fragment may remain intact orbecome further degraded, depending upon theenzyme and conditions used. Proteolytic IgG frag-mentation using three different enzymes is discussedbelow and summarized in Figure 20. Traditionally, proteolysis wasaccomplished in solution using free enzyme. Pierce has developedimmobilized enzyme products that enable better control of thedigestion and separation of reaction products from the enzyme.

Immobilized PapainPapain is a nonspecific, thiol-endopeptidase that has a sulfhydrylgroup in the active site, which must be in the reduced form foractivity. When IgG molecules are incubated with papain in thepresence of a reducing agent, one or more peptide bonds in thehinge region are split, producing three fragments of similar size:two Fab fragments and one Fc fragment.1 When Fc fragmentsare of interest, papain is the enzyme of choice because it yields a50,000 dalton Fc fragment.

Papain is primarily used to generate Fab fragments, but it also canbe used to generate F(ab′)2 fragments.2 To prepare F(ab′)2 fragments,the papain is first activated with 10 mM cysteine. The excesscysteine is then removed by gel filtration. If no cysteine is presentduring papain digestion, F(ab′)2 fragments can be generated. Thesefragments are often inconsistent, and reproducibility can be aproblem. If the cysteine is not completely removed, overdigestioncan be a problem.2

Figure 19. Structure of Fab, Fab′, F(ab′)2 and Fv Fragments.

Crystalline papain is often used for the digestion of IgG; however,it is prone to autodigestion. Mercuripapain, which is less proneto autodigestion than crystalline papain, can be used; however,both of these non-immobilized enzymes require an oxidant toterminate digestion. Immobilized papain is the preferred reagentbecause it allows for easy control of the digestion reaction, as wellas separation of enzyme from the crude digest. There is no needto develop an ion exchange method for separating the fragmentsfrom the enzyme. The use of immobilized papain will also preventformation of antibody-enzyme adducts, which can occur whenusing the soluble form of sulfhydryl proteases (such as papain).These adducts can be detrimental to fragments in the presenceof reductants.

Immobilization also increases stability of the enzyme against heatdenaturation and autolysis and results in longer maintenance ofactivity. Regeneration of the papain is often possible afterimmobilization, resulting in decreased costs. Cleavage can beregulated by digestion time or flow rate through a column, yieldingreproducible digests. Pierce Immobilized Papain (Product # 20341)offers all the advantages of immobilized enzyme supports (Figure 21).Pierce ImmunoPure® Fab Preparation Kit (Product # 44885) hasbeen optimized for human IgG digestions. It also has been usedsuccessfully for mouse and rabbit digestions, and suggestions onhow to vary the protocols for other species’ IgG are provided withthe kit. The procedure required that the IgG is able to be boundby Protein A, which is used to separate Fc from Fab fragments.

Figure 21. Preparation and isolation of Fab and Fc fragments with Immobilized Papain.

Figure 20. Use of papain, pepsin and ficin for IgG fragmentation.

F(ab′)2

(110,000 daltons)

Fc Fragments

Immobilized Ficin10 mM cysteine

Immobilized Papain

Fc (50,000 daltons)

2 Fab(50,000 daltons ea.)

IgG (150,000 daltons)

Immobilized Ficin1 mM cysteine

Immobilized Pepsin

HeavyChain

Preparation and Isolation of Fab and Fc Fragments

1. Equilibration of Immobilized Papain 2. Addition of IgG sample to Immobilized Papain

3. Digestion of IgG

Incubate in a shaker water bath at 37°C for the appropriate amount of time.Add IgG in 1.0 ml digestion

buffer to tube containing Immobilized Papain.

4. Separation of crude digest

Compress gel with separator and pour crude digest into clean tube.

5. Washing of Immobilized Papain 6. Addition of crude digest to Immobilized Protein A column

7. Collection of Fab fragments

a) Add 6 ml ImmunoPure® IgG Binding Buffer to Protein A column and collect eluate. Dialyze against PBS, pH 7.4 overnight.

b) Wash column with additional 3 ml binding buffer. Discard wash.

8. Collection of Fc and undigested IgG

a) Transfer Protein A column to another tube.

b) Elute Fc and undigested IgG from column with 6 ml ImmunoPure® IgG Elution Buffer.

Add crude digest to an equilibrated Immobilized Protein A Column. Collect column eluate.

a) Add 1.5 ml of Binding Buffer to gel as in A. Mix as in B.b) Add wash to rest of crude digest.

a) Add 0.5 ml of Immobilized Papain slurry + 4 ml of digestion buffer to the tube as in A.b) Mix the tube contents as in B.c) Compress the gel as in C and discard the buffer.d) Repeat steps a, b and c.e) Resuspend the gel in 0.5 ml buffer.

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Immobilized PepsinPepsin is a nonspecific endopeptidase that is active only at acid pH.It is irreversibly denatured at neutral or alkaline pH. Digestion bythe enzyme pepsin normally produces one F(ab′)2 fragment andnumerous small peptides of the Fc portion (Figure 20). Theresulting F(ab′)2 fragment is composed of two disulfide-connectedFab units. The Fc fragment is extensively degraded, and its smallfragments can be separated from F(ab′)2 by dialysis, gel filtrationor ion exchange chromatography.

F(ab′)2 can be separated by mild reduction into two sulfhydryl-containing, univalent Fab′ fragments. The advantage of Fab′fragments is that they can be conjugated to detectable labelsdirectly through their sulfhydryl groups, ensuring that theactive binding site remain unhindered and active. Pierce offers2-Mercaptoethylamine•HCl (2-MEA, Product # 20408) for mildreduction of F(ab′)2 fragments. For alternative labeling protocols,the free sulfhydryl may be blocked with an alkylating reagent,such as N-Ethylmaleimide (NEM, Product # 23030).

Immobilized Pepsin (Product # 20343) can be substituted for freepepsin in any application. Immobilized pepsin is advantageousbecause of its ability to immediately stop the digestion process,yielding reproducible digests. Immobilization of the enzyme allowsfor easy separation of the enzyme from the crude digest, eliminatingthe need to develop an ion exchange method for separating thefragments from the enzyme. Also, immobilization increases thestability of the pepsin against heat denaturation and autolysis,resulting in longer maintenance of activity. ImmunoPure® F(ab′)2

Preparation Kit (Product # 44888) has been optimized for humanIgG digestions. The kit also has been used successfully for rabbitand mouse IgG digestions. Suggestions on how to vary protocolsfor other species’ IgG (IgG must bind to Protein A) are provided.

Immobilized FicinFicin is a thiol protease that can digest mouse monoclonal IgG1 intoeither F(ab′)2 or Fab fragments, depending on the concentrationof cysteine used. Ficin will generate F(ab′)2 in the presence of 1 mMcysteine. Fab fragments will be generated with ficin in the presenceof 10 mM cysteine (Figure 20).

Ficin cleavage produces F(ab′)2 fragments of nearly identical size tothose obtained from IgG by pepsin but with immunoreactivities andaffinities comparable to those of intact IgG1 antibody.3 By increasingthe concentration of cysteine activator, Fab antigen-binding fragmentscan be generated.4 The integrity of the resultant antigen-bindingfragments is aided by the neutral pH conditions of the ficin digestion.The difficulties of using pepsin in this application makes ficindigestion the preferred method for producing F(ab′)2 fragmentsfrom murine monoclonal IgG1. Although F(ab′)2 fragments havebeen generated from an IgG1 antibody using preactivated papain,5stable, consistent product by papain is often difficult to obtain.6

Immobilized Ficin (Product # 44881) enables better control of thedigestion reaction than free ficin, resulting in antibody fragmentsthat are free of autodigestion products (Figure 22). In addition,the use of Immobilized Ficin eliminates the incorporation of ficininto antibody fragments. The ImmunoPure® IgG1 Fab and F(ab′)2

Preparation Kit (Product # 44880) was developed to allow gentleproduction and purification of both Fab and F(ab′)2 fragmentsfrom intact murine IgG1 antibodies. Immobilized Ficin can be usedrepeatedly to cleave an IgG1 subclass antibody, yielding either Fabor F(ab′)2 fragments. The type of fragment produced is controlledby the specific concentration of cysteine activator used duringthe digestion.

Figure 22. Selective preparation of monovalent or bivalent antigen-bindingfragments from IgG1. Figure 22A. Digestion products of mouse anti-avidinIgG1 monoclonal on immobilized ficin. 5 hours, 37°C, 10 mM cysteine. Ficin:Mab = 11:1 (mole:mole). Figure 22B. Size exclusion profile of fraction not boundto Protein A column. Component composition: 4% F(ab′)2, 96% Fab. TotalRecovery: 47% Fab. Figure 22C. Digestion products of mouse anti-avidinIgG1 monoclonal on immobilized ficin. 20 hours, 37°C, 1 mM cysteine. Ficin:MAB = 11:1 (mole:mole). Figure 22D. Size exclusion profile of fraction notbound to Protein A column. Component composition: 98% F(ab′)2, 2% Fab.Total Recovery: 35% F(ab′)2.

References1. Coulter, A. and Harris, R. (1983). J. Immunol. Methods 59, 199-203. 2. Goding, J. (1983). Monoclonal Antibodies: Principles and Practice. Academic Press

Inc., London, U.K. 3. Mariani, M., et al. (1991). Mol. Immunol. 28, 69-77.4. Sykaluk, L. (1992). Pierce Chemical Company, unpublished results.5. Buguslawski, S.J., et al. (1989). J. Immunol. Methods 120, 51-56.6. Milenic, D.E., et al. (1989). J. Immunol. Methods 120, 71-83.

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Table 10. Enzymes used for antibody digestion.

Molecular Ext. Coefficient pH Activators Imm. EnzymeEnzyme Weight A280 of 1% Type Specificity Optimum (Enhancers) Inhibitors (mg/ml BCA) pIPepsin 35,000 14.7 Acid 1 (1-5) 1 11

Papain 23,000 25 Thiol 6.5 (4-9.5) 9.6 1.5

Ficin 26,000 21 Thiol 6.5 (4-9.5) 1.5

Trypsin 24,000 14.3 Serine 8 10.5 1.5

*NBS = N-bromosuccinimide **DFP = diisopropyl fluorophosphate

organophosphorouscompounds, DFP**, benzimidine

Ca2+ acts as astabilizer

Arg, Lys

heavy metals, carbonyls, NEM,p -chloromercuro-benzoate

cysteine, sulfide,sulfite, cyanide,(EDTA) (NBS)(acridine dye)

Uncharged or aromaticamino acids

heavy metals, carbonyls, N-ethyl maleimide (NEM), p -chloromercuro-benzoate

cysteine, sulfide,sulfite, cyanide,(EDTA) (NBS*)(acridine dye)

Broad – preferArg, Lys, His,Gly, Tyr bonds

pH > 6, epoxidesBroad – preferPhe, Met Leu,Tryp bonds

Pierce has developed a complete kit to digest human or mouse IgGmolecules into Fab fragments and Fc fragments by using immobilizedpapain. After digestion, the fragments are purified on an immobi-lized Protein A column provided in the kit. Detailed instructionsallow for flexibility in the protocol for hard-to-digest antibodies.

Highlights of Fab Fragments:• Will not be affected by Fc receptors on cells such as

macrophages, B cells, T cells, neutrophils and mast cells• Will not precipitate antigen• Easier to make and purify than F(ab′)2 fragments• More rapid clearance of radiolabeled fragments from normal

tissue than whole IgG conjugates• Reduced immunogenicity (as a result of Fc region absence),

minimizes human anti-mouse immunoglobulin (HAMA) response• Fragments are less susceptible to phagocytosis

Antibody Fragment Applications:• Immunohistochemistry• Immunoassays, including in vitro diagnostic assays• Radioimmunolocalization for tumor detection and radiotherapy• Immunotargeting through the use of fragment conjugates as

immunotoxins• Crystallographic study of antibody-binding sites• Study of Fc-binding proteins and effector functions

Highlights of Fc Fragments:• Useful for studying effector functions of IgG without interference

from antigen-binding sites• Fc fragments can be used as blocking agents for histochemical

staining

ReferencesCoulter, A. and Harris, R. (1983). J. Immunol. Methods 59, 199-203.Drewett, J.G., et al. (1995). J. Biol. Chem. 270, 4668-4674.Mage, M.G. (1980). Methods Enzymol. 70, 142-150.Milenic, D.E., et al. (1989). J. Immunol. Methods 120, 71-83.Rousseaux, J., et al. (1983). J. Immunol. Methods 64, 141-146.Schmalzing, D., et al. (1995). Anal. Chem. 67(3), 606-612.Wolf, A., et al. (1994). J. Biol. Chem. 269, 23051-23058.

Ordering InformationU.S.

Product # Description Pkg. Size Price44885 ImmunoPure® Fab Preparation Kit Kit $425

Isolates and purifies Fab fragments from up to 200 mg of mouse IgG.Includes: Immobilized Papain 5 ml

Cysteine•HCl 1 gAffinityPak™ Immobilized 2 x 2.5 mlProtein A Columns

ImmunoPure® IgG Binding Buffer 1,000 mlImmunoPure® IgG Elution Buffer 500 ml

20341 Immobilized Papain 5 ml $148Support: Cross-linked 6% beaded agaroseActivity: 7 BAEE units per ml of settled gelLoading: 250 µg/ml of gel

ImmunoPure® Fab Preparation KitThe easiest, most convenient way to generate Fab fragments from IgG.

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46800-874-3723 • 815-968-0747 • www.piercenet.com

Problems with digesting mouse monoclonal IgG1 can now beovercome by using immobilized Ficin. Ficin cleavage producesF(ab′)2 fragments of nearly identical size to those obtained fromIgG by pepsin, but with immunoreactivities and affinities compa-rable to those of the intact IgG1 antibody. Similarly, by increasingthe concentration of cysteine activator in the digestion buffer,Fab fragments can be created from the original IgG.

Highlights:• Can generate both Fab and F(ab′)2 fragments from mouse IgG1

• Reaction can be easily controlled• Antibody fragments are free of autodigestion products• Ficin contamination into antibody fragments is eliminated

ReferencesKurkela, R., et al. (1988). J. Immunol. 110, 229-236.Mariani, M., et al. (1991). Mol. Immunol. 28, 69-77.Sykaluk, L. (1992). Pierce Chemical Company, unpublished results.Wilson, K.M., et al. (1991). J. Immunol. Methods 138, 111-119.

Ordering InformationU.S.

Product # Description Pkg. Size Price44880 ImmunoPure® IgG1 Fab and F(ab′)2 Kit $466

Preparation KitFor digesting up to 250 mg of mouse IgG1.Includes: Immobilized Ficin Columns 5 x 2 ml

(Activity = 1.2 mg/ml gel)Cysteine•HCl 1 gImmunoPure® IgG1 Digestion 50 mlBuffer (10X)AffinityPak™ Immobilized 2 x 2.5 mlProtein A Columns

ImmunoPure® IgG Binding Buffer 1,000 mlImmunoPure® IgG1 Mild 2 x 500 mlElution Buffer

Immobilized Ficin Storage Buffer 100 mlColumn Extenders 2

44881 Immobilized Ficin 5 ml $14144889 Cysteine•HCl 5 g $ 37

Pierce offers a complete kit for digesting antibodies into F(ab′)2

fragments that retain antigen-binding activity. Using immobilizedpepsin allows the antibody digest to be free of any enzyme contam-inants. Purifying F(ab′)2 fragments is as easy as passing the solutionover an immobilized protein A column. Detailed instructions allowfor flexibility in the protocol for hard-to-digest antibodies.

Highlights of F(ab′)2 Fragments:• Will not be affected by Fc receptors on cells such as

macrophages, B cells, T cells, neutrophils and mast cells• They are divalent, which is recommended for retaining antigen-

binding capabilities of low affinity antibodies• Will precipitate antigen

ReferencesKulkarni, P.N., et al. (1985). Cancer Immunol. Immunother. 19, 211-214.Lamoyi, E. (1986). Methods Enzymol. 121, 652-663.Pruimboom, I.M., et al. (1999). Infect. Immun. 67(3), 1292-1296.Rousseaux, J., et al. (1983). J. Immunol. Methods 64, 141-146.Wedrychowski, A., et al. (1993). Biotechnology (N-Y) 11, 486-489.

Ordering InformationU.S.

Product # Description Pkg. Size Price44888 ImmunoPure® F(ab′)2 Preparation Kit Kit $425

Digests up to 200 mg of mouse IgG.Includes: AffinityPak™ Immobilized 2 x 2.5 ml

Protein A ColumnsImmunoPure® IgG Binding Buffer 1,000 mlImmunoPure® IgG Elution Buffer 500 mlImmobilized Pepsin 5 ml

20343 Immobilized Pepsin 5 ml $149Support: Cross-linked 6% beaded agaroseActivity: ≥ 2,000 units per ml of settled gelLoading: 2-3 mg/ml of gel

ImmunoPure® IgG1 Fab and F(ab′)2 Preparation KitGenerate both Fab and F(ab′)2 fragments from monoclonal IgG1 antibodies.

ImmunoPure® F(ab′)2 Preparation KitThe easiest, most convenient way to generate F(ab′)2 fragments from IgG.

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Fragmentation of IgMIgM is an extremely large molecule that has a tendency to interactwith other molecules and matrices besides the antigen. The largesize of IgM creates difficulties in applications in which IgM is usedfor in vitro experiments. Intact IgM does not effectively penetratetissues for immunohistochemical studies; it is necessary toproduce smaller, active fragments for these studies. Also, becauseIgM molecules have difficulty permeating cell membranes, theyare not ideal for use in vivo. Fragments are cleared more rapidlythan intact IgM.

Each species of IgM reacts differently to enzymatic cleavage andreduction. For example, the relative structure of mouse and humanIgM differ in the manner in which the monomers are linked to givethe pentameric form, primarily as a result of differences in thelocation of disulfides between the monomers.1 Oligosaccharidescomponents, which may hinder enzymatic cleavage, also varybetween species. Therefore, optimal digestion and reductionconditions for one species may prove ineffective for another.

Fragmentation of IgM by proteolyic enzymes proceeds differentlyfrom IgG fragmentation. These changes are related to differencesin structure. The heavy (µ) chains are folded into multiple globulardomains, and IgM has an actual domain (Cµ2) in place of a hingeregion. Cµ2 lacks the proline-rich sequence that is found in thehinge region of IgG. This proline sequence makes the hinge moresusceptible to cleavage. Also, IgM has a large carbohydrateportion in the Cµ2, which may sterically interfere with the actionof proteolytic enzymes.

Enzymes and Reagents for IgM FragmentationPapain is a nonspecific, thiol-endopeptidase that has a sulfhydrylgroup in the active site, which must be in the reduced form foractivity. Papain has been shown to produce heterogeneousfragments from IgM. Oligosaccharides in the hinge region ofIgM interfere with papain digestion, causing a cleavage shift of3-5 amino acids in either direction.

Pepsin is a nonspecific endopeptidase that is active only at anacid pH, and it is irreversibly denatured at neutral or alkaline pH.It is possible to produce F(ab′)2, Fab and Fv fragments usingpepsin to digest IgM (Figure 23). Many methods have beendeveloped that use pepsin to produce different IgM fragmentsfrom different species.2

Figure 23. Fragmentation of IgM.

Trypsin is a serine protease that reacts optimally at pH 8.0. Ingeneral, increasing the enzyme/substrate ratio and/or temperaturewill increase the rate of digestion. Trypsin can generate F(ab′)2,Fab, “IgG”-type and Fc5µ fragments from IgM (Figure 23). Trypsindigestion of several species of IgM was studied using trypsin withand without urea pretreatment.2 Urea alters the susceptibility ofthe domains to digestion and produces different fragments thanthose digested in aqueous buffer. Many other procedures havebeen developed to digest IgM using trypsin.3

Mild reduction can be achieved using 2-Mercaptoethylamine•HCl(2-MEA, Product # 20408). Reduction will vary among IgMspecies, but an “IgG”-type and/or reduced IgG (“rIgG”) shouldbe formed in varied proportions, depending upon reduction timeand/or temperature (Figure 23).4 Fragmentation of mouse IgMalso produces an inverted “IgG”-type fragment.

“IgG” type-M(200,000 daltons)

Inverted “IgG” (Mouse)(200,000 daltons)

Fc 5m-H(340,000 daltons)

F(ab′)2(150,000 daltons)

Fab-H(45,000 daltons)

“IgG” type(200,000 daltons)

“r IgG” (110,000 daltons)

F(ab′)2(150,000 daltons)

Fab (45,000 daltons)

Fv(25,000 daltons)

IgM

Immobilized

Trypsin

2-MEA•HCI

Imm

obilized Pepsin

H=human onlyM=mouse only

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IgM Fragmentation KitPierce has developed the ImmunoPure® IgM Fragmentation Kit(Product # 44887) to allow quick, easy production of fragmentsfrom mouse and human IgM. This kit can be used for speciesother than human and mouse; however, the protocols have notbeen optimized for all species. The kit contains everything neededto produce IgM fragments using trypsin, pepsin and 2-MEAprotocols. The trypsin and pepsin are supplied in immobilizedforms, eliminating the need to separate enzyme from the IgMfragments. The IgM is digested as it passes through the prepackedimmobilized enzyme columns. The enzyme remains bound to thesupport matrix, ensuring there is no autodigested enzyme tocontaminate the IgM fragments. Also, the immobilized enzymesoffer increased stability against heat denaturation and autolysis.Regeneration of the immobilized enzymes makes the process

more cost-efficient. The cleavage of IgM is easily regulated byadjustment of incubation times. Sample concentrators areincluded in the kit for easy fragment separation and concentra-tion. Complete instructions and protocols are also included.

References1. Milstein, C.P., et al. (1975). Biochem. J. 151, 615-624.2. Beale, D. and Van Dort, T. (1982). Comp. Biochem. Physiol. 71B(3), 475-482.3. Plaut, A.G. and Tomasi, Jr., T.B. (1970). Proc. Natl. Acad. Sci. USA 65(2), 318-322.4. Bevan, M.J., et al. (1972). Progr. Biophys. Molec. Biol. 25, 131.

The large size of IgM creates difficulties in applications in whichIgM is used for in vitro experiments. Intact IgM does not effectivelypenetrate tissues for immunohistochemical studies, therefore itis necessary to produce smaller, active fragments for in vitro orin vivo studies. Pierce created a kit combining immobilizedtrypsin and pepsin to easily generate a variety of IgM fragments(Figure 23).

Highlights:• Immobilized trypsin can generate F(ab′)2, Fab, “IgG”-type and

Fc(5µ) fragments from IgM• Immobilized pepsin can produce F(ab′)2, Fab and Fv fragments

from IgM• Complete kit, including detailed instructions, to digest and

purify IgM fragments• Immobilized enzymes prevent enzyme contaminants in final

fragment preparation

ReferencesBeale, D. and Van Dort, T. (1982). Comp. Biochem. Physiol. [B] 71(3), 475-482.Chen, F.-M. and Epstein, A.L. (1988). Antibody, Immunoconjugates, andRadiopharmaceuticals 1(4), 333-341.Herremans, T., et al. (2000). Clin. Diagn. Lab Immunol. 7(1), 40-44.Kakimoto, K. and Onoue, K. (1974). J. Immunol. 112(4), 1373-1382.Lin, L.-C. and Putnam, F.W. (1978). Proc. Natl. Acad. Sci. USA 75(6), 2649-2653.Poncet, P., et al. (1988). Mol. Immunol. 25(10), 981-989.

Ordering InformationU.S.

Product # Description Pkg. Size Price44887 ImmunoPure® IgM Fragmentation Kit Kit $610

Fragments up to 26 mg of human or mouse IgM.Includes: Immobilized Trypsin Columns 2 x 2 ml

Immobilized Pepsin Columns 2 x 2 ml2-Mercaptoethylamine 6 mgIgM Digestion Buffer 400 mlIgM F(ab′)2 Digestion Buffer 200 mlIodoacetamideSample ConcentratorsDesalting Columns 2 x 5 ml

20343 Immobilized Pepsin 5 ml $14920408 2-Mercaptoethylamine•HCI 6 x 6 mg $117

ImmunoPure® IgM Fragmentation KitMakes IgM fragmentation easy!

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AntibodyFragmentation

Structure courtesy of: Title: Chicken Egg White Cystatin Wildtype, NMR, 16 Structures; PDB: 1A67; Citation: Dieckmann, T., Mitschang, L., Hofmann, M., Kos, J., Turk, V., Auerswald, E. A., Jaenicke, R., Oschkinat, H.:The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution. J Mol Biol 234 pp. 1048 (1993).

Antibody Modification SitesUnderstanding the functional groups available on an antibody isthe 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 the entireantibody.Sulfhydryl groups (–SH) are found on cysteine residues and areformed by selectively reducing disulfide bonds in the hingeregion of the antibody.Carbohydrate residues containing cis-diols can be oxidized(–CHO) to create active aldehydes for coupling. These arelocalized to the Fc region on antibodies and are more abundanton polyclonal antibodies.

Figure 24. Antibody labeling sites.

Most antibody labeling strategies use one of three targets(Figure 24). The most common target for antibody labeling isprimary amines, which are found primarily on lysine residues.They are abundant, widely distributed and easily modified due totheir reactivity and their location on the surface of the antibody.The lone drawback to this labeling strategy is that it often resultsin a significant decrease in the antigen-binding activity of theantibody. The decrease may be particularly pronounced whenworking with monoclonal antibodies or when multiple labels areattached to the antibody.

The second common target for labeling antibodies is carbohydratemoieties because antibodies are often significantly glycosylated.Because the glycosylation sites are predominantly found on theFc portion of the antibody, they can often be modified withoutsignificantly reducing the antigen-binding capacity. Labeling carbo-hydrates requires more steps than labeling amines because thecarbohydrates must first be oxidized to create reactive aldehydes;however, it generally results in antibody conjugates with high activity.

The third common target is sulfhydryls that exist in proteins underreducing conditions, but more often are found in oxidized formas disulfide bonds. Disulfide bonds are important contributors toantibody structure as they participate in the tertiary structure ofeach subunit, covalently connecting the heavy and light chainsand connecting the two halves of an antibody in the hinge region.These disulfides in the hinge region are the primary target forsulfhydryl labeling of an antibody because they are easily reducedto sulfhydryls, splitting the antibody into monovalent halves(rIgG) without damaging the antigen-binding sites. Like carbo-hydrates, they require more steps to label yet they result inhigh-activity conjugates.

Antigen-bindingsite Light Chains

Carbohydrate Carbohydrate

HingeRegion

CH2 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 Heavy Chains

VH

Amines onlysine residues

Sulfhydryls created whenantibody is reduced

NH2

NH2

NH2

NH2

OverviewAntibodies, like other proteins, can be cova-

lently modified in many ways to suit the purpose of a

particular assay. Many immunological methods involve the

use of labeled antibodies, and a variety of reagents have been

created to allow labeling of antibodies. Enzymes,biotin, fluorophores and

radioactive isotopes are all commonly used to provide a detection signal in

biological assays. Covalently attaching such a label to an antibody combines the

unique specificity of the antibody with a sensitive means for detection, thus creating an

ideal probe molecule. Aside from antibodies, these same labels can be attached to avidin,

streptavidin, Fc-binding proteins such as Protein A or G, and many other proteins.

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Enzyme LabelingEnzymes offer the advantages of long shelf life, high sensitivity andthe possibility of direct visualization. The disadvantages of enzymesas labels include multiple assay steps, some hazardous substratesand the possibility of interference from endogenous enzymes.Enzymes also may give poor resolution in cytochemistry. The useof enzyme labels is recommended for immunohistochemistry,immunoblots and quantitative and qualitative immunoassays.

Using enzymes as labels offers several advantages over fluores-cently labeled and radiolabeled substances. Enzyme immunoassayreagents are more stable and do not have the dangers associatedwith radioisotopic labels. In addition, enzyme assays can be atleast as sensitive as radioimmunoassays. Many enzyme detectionmethods are visual or use a standard spectrophotometer,eliminating the need for expensive, sophisticated equipment.

When detecting cellular antigens to tissue structures, enzymelabels also have advantages. Antibodies (and especially theirfragments) conjugated to small enzymes can readily cross cellmembranes. It is then possible to localize and observe cellularantigens to tissue structures using light microscopy. In addition,tissue sections that have been developed with the appropriatesubstrate can be stable for years. This is in stark contrast to mostimmunofluorescent staining techniques, in which the fluorescentsignal rapidly decreases upon exposure to light.

Selectionof an enzymeand the appropriatesubstrate solution aredependent on the applicationinvolved (Table 11). Enzyme-labeled antibodies perform well inimmunoblotting, histochemicalstaining and ELISA. They can providean instant visual result and good sensitivity;however, the rate of the enzyme reaction must be measuredto obtain an accurate indication of the amount of bound enzyme,making them difficult to use in quantitative assays. In addition,enzyme-labeled reagents are not homogeneous. In immunohis-tochemistry, nonspecific staining can present a problem whenusing enzymes; additional problems include endogenousperoxidase or phosphatase activity. In these cases, a differentenzyme may need to be selected. Table 11 compares peroxidaseand alkaline phosphatase, along with their respective advantages,disadvantages and recommended applications.

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Table 11. Comparison of horseradish peroxidase and alkaline phosphatase uses.

RecommendedEnzyme Advantages Disadvantages Substrate Signal ApplicationsHorseradish Peroxidase

Alkaline Phosphatase

Western blottingInsoluble-NBT/BCIP,Fast Red TR/AS-MX

ELISASoluble-PNPPEndogenous activities –some in tissuesLarge conjugates

Good substrates for Western blotting,ELISA and immunohistochemistry

Western blottingImmunohistochemistry

Insoluble-Chloronaphthol,DAB/cobalt, TMB-blotting,SuperSignal® West Substrate

ELISASoluble-TMB, ABTS, OPDQuantaBlu™ Fluorogenic SubstrateSuperSignal® ELISA Substrate

High endogenous levelsin blood cells

Good substrates for Western blotting,ELISA, immunohistochemistry and1:1 conjugatesFast signal generation

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

Alkaline phosphatase, a 140 kDa protein that is generally isolatedfrom calf intestine, catalyzes the hydrolysis of phosphate groupsfrom a substrate molecule, resulting in a colored or fluorescentproduct or the release of light as a byproduct. AP has optimalenzymatic activity at a basic pH (pH 8-10) and can be inhibitedby cyanides, arsenate, inorganic phosphate and divalent cationchelators, such as EDTA. As a label for Western blotting, AP offersa distinct advantage over other enzymes. Because its reactionrate remains linear, detection sensitivity can be improved bysimply allowing a reaction to proceed for a longer time period.

Horseradish peroxidase is a 40 kDa protein that catalyzes theoxidation of substrates by hydrogen peroxide, resulting in acolored or fluorescent product or the release of light as a byproduct.HRP functions optimally at a near-neutral pH and can be inhibitedby cyanides, sulfides and azides. Antibody-HRP conjugates aresuperior to antibody-AP conjugates with respect to the specificactivities of both the enzyme and antibody. In addition, its highturnover rate, good stability, low cost and wide availability ofsubstrates make HRP the enzyme of choice for most applications.

Table 12. Comparison of horseradish peroxidase and alkaline phosphatase physical properties.

Horseradish Peroxidase Alkaline PhosphataseSize 40 kDa 140 kDaPrice Relatively Inexpensive Relatively ExpensiveStability (Storage) Stable at < 0˚C Unstable at < 0˚CNumber of Substrates Many FewKinetics Rapid SlowerpH optimum 5-7 8-10

Maleimide activationThe heterobifunctional cross-linker SMCC (Product # 22360) andits water-soluble analog Sulfo-SMCC (Product # 22322) havemore general utility in preparing immunologically active HRP orAP conjugates. They are most useful when preparing conjugatesof reduced IgG and F(ab′)2 because these methods involve the initialstep of preparing a maleimide-activated (sulfhydryl-reactive)enzyme derivative. Studies have shown that the two-step maleimidemethod is superior to glutaraldehyde or metaperiodate methodsfor enzyme conjugation (Figure 25). The maleimide method giveshigher yields with less polymerization, producing a conjugatepreparation with superior immunoassay characteristics.

Maleimide-activated enzymes can be prepared using the heter-obifunctional cross-linker Sulfo-SMCC. This reagent contains anN -hydroxy-sulfosuccinimide (Sulfo-NHS) functional group anda maleimide functional group and it is water-soluble due to thepresence of the sulfonate (–SO3

-) group on the N-hydroxysuc-cinimide 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-SMCC showed that it is less prone to hydrolysis to the maleamicacid than the non-sulfonated SMCC. The maleimide groups ofSulfo-SMCC exhibit no decomposition at pH 7 at 30˚C within 6hours. The Sulfo-NHS ester group reacts with primary amines onthe enzyme surface to form a stable amide bond. After this firststep of conjugation, the enzyme will have maleimide groups on itssurface that react optimally toward sulfhydryl groups betweenpH 6.5 and 7.5 to form stable thioether bonds. Maleimide-mediated conjugation strategies are summarized in Figure 25.

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Figure 25. Three strategies for maleimide-mediated conjugation of enzymes.

SH

Three methods for free sulfhydryl generation Maleimide activation of enzyme

1. Native protein has a free sulfhydryl on its surface.

3. 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

2.MEAEDTA

NH2SATA

CH2 S

O

C CH3H

CH2 SH

SH

H

N

H

N

O

C

O

O

OO

OO

N

SMCC

NHS

OH

O

O

N

N + H2N

S

O

ON N

O

E

E

E

O

ON

O N

S S

Protein

ProteinProtein

Protein

Protein

Protein

Protein

SH SH

Protein

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 and Radiopharmaceuticals 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.

Ordering InformationU.S.

Product # Description Pkg. Size Price31490 ImmunoPure® Horseradish Peroxidase 10 mg $ 5331491 ImmunoPure® Horseradish Peroxidase 100 mg $ 190* SuperSignal ® Technology is protected by U.S. patent # 6,432,662.

Horseradish PeroxidaseIts high-specific enzyme activity makes it the enzyme of choice.

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Antibody Labeling

Two reagents, Mercaptoethylamine•HCl (Product # 20408) andSATA (Product # 26102), are available to produce free sulfhydrylson macromolecules for conjugation to the maleimide-activatedenzymes. For labeling antibody molecules, mild reduction withMercaptoethylamine•HCl (MEA) results in two half-antibodyfragments containing free sulfhydryl groups in the hinge region.Labeling in this area is advantageous because it directs themodification away from the antigen-binding region. Nativeproteins lacking a free sulfhydryl on their surface can be reactedwith SATA to generate blocked sulfhydryl groups. The SATAmolecule reacts with primary amines via its NHS ester end toform stable amide linkages. The acetylated sulfhydryl group(blocked) is stable until treated with hydroxylamine to generatethe 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 the firststep of the two-step maleimide method, simplifying and facilitatingthe conjugation protocol, while saving several hours. They canbe used to prepare enzyme conjugates directly from proteins,peptides or other ligands that contain a free -SH group. Tworeagents, SATA and mercaptoethylamine•HCl, are also includedin the kit formats to produce free sulfhydryls on macromoleculesfor conjugation.

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

Ordering InformationU.S.

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 PhosphataseConjugation Buffer 20 mlBupH™ Tris Buffered Saline 2 packsBupH™ Phosphate Buffered Saline 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 PeroxidaseConjugation Buffer 20 ml2-Mercaptoethylamine•HCl 6 mgSATA 2 mgDimethylformamide 1 mlHydroxylamine•HCl 5 mgPolyacrylamide Desalting Column 1 x 10 ml

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 Mdiethanolamine, 0.5 mM MgCl2, pH 7.8

Specific Activity per mg Protein

Buffer 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.

Ordering InformationU.S.

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

Alkaline PhosphataseA highly sensitive enzyme for ELISA and immunohistochemical applications.

EZ-Link® Maleimide Activated Alkaline Phosphatase and Horseradish PeroxidaseMake quick and easy enzyme conjugates.

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PeriodateGlycoproteins such as HRP, glucose oxidase and most antibodymolecules can be activated for conjugation by treatment withperiodate. Oxidizing polysaccharide residues in a glycoprotein withsodium periodate provides a mild and efficient way of generatingreactive aldehyde groups for subsequent conjugation with amine-or hydrazide-containing molecules via reductive amination(Figure 26). Some selectivity of monosaccharide oxidation maybe accomplished by regulating the concentration of periodate inthe reaction medium. In the presence of 1 mM sodium periodate,sialic acid groups are specifically oxidized at adjacent hydroxylresidues, cleaving off two molecules of formaldehyde and leavingone aldehyde group. At higher concentrations of sodium periodate(10 mM or greater), other sugar residues will be oxidized at pointswhere adjacent carbon atoms contain hydroxyl groups. Thisreaction should be performed in the dark to prevent periodatebreakdown and for a limited period of time (15-30 minutes) toavoid loss of enzymatic activity.

Figure 26. Conjugation scheme for periodate oxidation and subsequentreductive amination.

Cross-linking with an amine-containing protein takes placeunder alkaline pH conditions through the formation of Schiffbase intermediates. These relatively labile intermediates can bestabilized by reduction to a secondary amine linkage with sodiumcyanoborohydride. Reductive amination has been done usingsodium 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 controlled toaffect the efficiency of the cross-linking process and the size of theresultant antibody-enzyme complexes formed. At physiological pH,

the initial Schiff base formation is less efficient and conjugates oflower molecular weight result. At more alkaline pH (i.e., pH 9-10),Schiff base formation occurs rapidly and with high efficiency,resulting in conjugates of higher molecular weight and greaterincorporation of enzyme when oxidized enzyme is reacted inexcess. Low molecular weight conjugates may be more optimalfor immunohistochemical staining or blotting techniques in whichpenetration of the complex through membrane barriers is animportant consideration. Washing steps also more effectivelyremove excess reagent if the conjugate is of low molecular weight,thus maintaining low background in an assay. By contrast,conjugates of high molecular weight are more appropriate forELISA procedures in a microplate format, where high sensitivityis important and washing off excess conjugate 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.

Ordering InformationU.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

31496 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 $317Antibody 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

20504 Sodium Meta-Periodate 25 mg $ 22

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

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