Hermanson G. Bioconjugate Techniques, Second Edition

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800 20. Antibody Modiﬁ cation and Conjugation

Two-Step Glutaraldehyde Protocol

1. Dissolve the enzyme at a concentration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M

NaCl, pH 6.8.

2. Add glutaraldehyde to a ﬁ nal concentration of 1.25 percent.

3. React overnight at room temperature.

4. Purify the activated enzyme from excess glutaraldehyde by gel ﬁ ltration using a desalting

resin or by dialysis against PBS, pH 6.8.

5. Dissolve the antibody to be conjugated at a concentration of 10 mg/ml in 0.5 M sodium

carbonate, pH 9.5. Mix the activated enzyme with the antibody at the desired molar

ratio to effect the conjugation. Mixing the equivalent of 4 mg of enzyme per mg of anti-

body usually results in acceptable conjugates.

6. React overnight at 4 °C.

7. To reduce the resultant Schiff bases and any excess aldehydes, add sodium borohydride

to a ﬁ nal concentration of 10 mg/ml.

Note: Some protocols avoid a reduction step. As an alternative to reduction, add 50  l

of 0.2 M lysine in 0.5 M sodium carbonate, pH 9.5 to each ml of the conjugation reac-

tion to block excess reactive sites. Block for 2 hours at room temperature. Other amine-

containing small molecules may be substituted for lysine—such as glycine, Tris buffer, or

ethanolamine.

8. Reduce for 1 hour at 4 °C.

9. To remove any insoluble polymers that may have formed, centrifuge the conjugate or

ﬁ lter it through a 0.45 m ﬁ lter. Purify the conjugate by gel ﬁ ltration or dialysis using

PBS, pH 7.4.

1.3. Reductive Amination-Mediated Conjugation

Oxidation of polysaccharide residues in glycoproteins with sodium periodate provides an efﬁ -

cient way of generating reactive aldehyde groups for subsequent conjugation with amine- or

hydrazide-containing molecules via reductive amination (Chapter 1, Section 4.4, and Chapter

2, Section 5.3). Some selectivity of monosaccharide oxidation may be accomplished by regulat-

ing the concentration of periodate in the reaction medium. In the presence of 1 mM sodium

periodate at approximately 0 °C sialic acid groups will be speciﬁ cally oxidized at their adja-

cent hydroxyl residues on the Nos. 7, 8, and 9 carbon atoms, cleaving off two molecules of

formaldehyde and leaving one aldehyde group on the No. 7 carbon. At higher concentrations

of sodium periodate (10 mM or greater) at room temperature other sugar residues will be oxi-

dized at points where adjacent carbon atoms contain hydroxyl groups.

Thus, glycoproteins such as HRP, GO, or most antibody molecules can be activated for

conjugation by brief treatment with periodate. Crosslinking with an amine-containing protein

takes place under alkaline pH conditions through the formation of Schiff base intermediates.

These relatively labile intermediates can be stabilized by reduction to a secondary amine link-

age with sodium cyanoborohydride ( Figure 20.8 ).

The use of periodate coupling chemistry for HRP ﬁ rst was introduced by Nakane and Kawaoi

(1974; see also Nakane, 1975). In the ﬁ rst step of their protocol, the few amine groups on

HRP were initially blocked with 2,4-dinitroﬂ uorobenzene (DNFB) before periodate oxidation.

The blocking step was designed to eliminate the possibility of self-conjugation of enzyme mol-

ecules during reductive amination with an immunoglobulin. However, Boorsma and Streefkerk

(1976a, b) determined that HRP still can dimerize even after DNFB blocking, perhaps by a

mechanism similar to Mannich condensation (Chapter 2, Section 6.2) or through aldol for-

mation. In fact, amine-blocked, periodate-oxidized HRP will form insoluble complexes during

storage after just weeks in solution at room temperature or 4 °C, indicating that another route

of conjugation is taking place.

Reductive amination crosslinking has been done using sodium borohydride or sodium

cyanoborohydride; however cyanoborohydride is the better choice since it is more speciﬁ c for

reducing Schiff bases and will not reduce aldehydes. Small blocking agents such as lysine, gly-

cine, ethanolamine, or Tris can be added after conjugation to quench any unreacted aldehyde

sites (Mannik and Downey, 1973; Barbour, 1976; Mattiasson and Nilsson, 1977). Ethanolamine

and Tris are the best choices for blocking agents, since they contain hydrophilic hydroxyl

groups with no charged functional groups.

1. Preparation of Antibody–Enzyme Conjugates 801

Figure 20.8 Enzymes that are glycoproteins like HRP may be oxidized with sodium periodate to produce reac-

tive aldehyde residues. Conjugation with an antibody then may be done by reductive animation using sodium

cyanoborohydride.

802 20. Antibody Modiﬁ cation and Conjugation

The pH of the reductive amination reaction can be controlled to affect the efﬁ ciency of the

crosslinking process and the size of the resultant antibody–enzyme complexes formed. At phys-

iological pH, the initial Schiff base formation is less efﬁ cient and conjugates of lower molecular

weight will result. At more alkaline pH values (i.e., pH 9–10), Schiff base formation occurs

rapidly and with high efﬁ ciency, resulting in conjugates of higher molecular weight and greater

incorporation of enzyme (when oxidized HRP is reacted in excess).

The ability to select the relative size of the antibody–enzyme complex is important depending

on the assay application. Low-molecular-weight conjugates may be more optimal for immuno-

histochemical staining or blotting techniques where penetration of the complex through mem-

brane barriers is an important consideration. Washing steps also more effectively remove excess

reagent if the conjugate is of low molecular weight, thus maintaining a low background signal

in an assay. By contrast, conjugates of high molecular weight are more appropriate for ELISA

procedures in a microplate or array format, where high sensitivity is important, but washing

off excess conjugate is not a problem.

The protocols appearing in the literature vary according to the amount of periodate used

during polysaccharide oxidation, the type of reductant and blocking agent employed for reduc-

tive amination, and the pH at which the various reactions are done. This variability indicates

considerable ﬂ exibility in the protocols, all of which yield usable antibody–enzyme conjugates.

There are, however, several conclusions that can be drawn from these studies: Investigations

done using HRP indicate the optimal concentration of sodium periodate during oxidation to be

approximately 4–8 mM (Tussen and Kurstak, 1984). This reaction should be performed in the

dark to prevent periodate breakdown and for a limited period of time (no more than 15–30 min-

utes) to avoid loss of enzymatic activity. The conjugation reaction should be done at alkaline pH

(7.2–9.5) in the presence of a reducing agent to stabilize the Schiff base intermediates. If sodium

cyanoborohydride is used as the reductant, a blocking agent should be added at the completion

of the conjugation reaction to cap excess aldehyde sites. The following protocol follows these

general guidelines and works well especially in the preparation of HRP–antibody conjugates.

Activation of Enzymes with Sodium Periodate

Enzymes that are glycosylated (i.e., HRP and GO) may be oxidized according to the following

method to produce aldehyde groups for reductive amination coupling to an antibody molecule.

Protocol

1. Dissolve the enzyme to be oxidized in water or 0.01 M sodium phosphate, 0.15 M NaCl,

pH 7.2, at a concentration of 10–20 mg/ml.

2. Dissolve sodium periodate in water at a concentration of 0.088 M. Protect from light.

3. Immediately add 100 l of the sodium periodate solution to each ml of the enzyme solu-

tion. This ratio of addition results in an 8 mM periodate concentration in the reaction

mixture. Mix to dissolve. Protect from light.

4. React in the dark for 15–20 minutes at room temperature. If HRP is the enzyme being

oxidized, a color change will be apparent as the reaction proceeds—changing the brown-

ish/gold color of concentrated HRP to green. Limiting the time of oxidation will help to

preserve enzyme activity.

5. Immediately quench the reaction by the addition of 0.1 ml of glycerol per ml of reac-

tion solution. Instead of glycerol, N-acetylmethionine may be added to quench the reac-

tion, because the thioether of the methionine side chain will react with periodate to form

sulfoxide or sulfone products (Geoghegan and Stroh, 1992). In addition, sodium sulﬁ te

(Na

) was used by Stolowitz et al. (2001) to quench the periodate oxidation of HRP

in solution. This may be the simplest route to stopping the reaction, as sulﬁ te is inex-

pensive and the reduction doesn ’t form reactive by-products. Add quenching reagent

to provide at least a 2  molar excess over the amount of periodate initially added to

the reaction. Alternatively, the reaction may be stopped by immediate gel ﬁ ltration on a

desalting resin. If a dextran-based resin is used for the chromatography, the support itself

will react with sodium periodate to quench excess reagent. Purify the oxidized enzyme by

gel ﬁ ltration using 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. To obtain efﬁ cient

separation between the oxidized enzyme and excess periodate (or quenching agent), the

sample size applied to the column should be at a ratio of no more than 5 percent sample

volume to the total column volume. Collect 0.5 ml fractions and monitor for protein at

280 nm. HRP also may be detected by its absorbance at 403 nm. When oxidizing large

quantities of HRP, the fraction collection process may be done visually—just pooling the

main colored HRP peak as it comes off the column.

6. Pool the fractions containing protein. Adjust the enzyme concentration to 10 mg/ml for

the conjugation step (see next section). The periodate-activated enzyme may be stored

frozen or freeze-dried for extended periods without loss of activity. Do not store the

preparation in solution at room temperature or 4 °C, since precipitation will occur over

time due to self-polymerization.

Activation of Antibodies with Sodium Periodate

Many immunoglobulin molecules are glycoproteins that can be periodate-oxidized to con-

tain reactive aldehyde residues. Polyclonal IgG molecules often contain carbohydrate in the

Fc portion of the molecule. This is sufﬁ ciently removed from the antigen binding sites to allow

conjugation to take place through the polysaccharide chains without compromising activity.

Occasionally, however, some antibodies may contain sites of glycosylation near the antigen

binding regions, and in this situation conjugation through these sites may affect binding activ-

ity. Although antibody–enzyme conjugation by reductive amination typically is done by oxida-

tion of the enzyme with subsequent crosslinking to an amine-containing antibody, oxidation of

the antibody with subsequent conjugation to an amine- or hydrazide-containing molecule also

is possible. It should be noted, however, that many monoclonal antibodies are not glycosylated

and therefore cannot be used in this protocol. Recombinant antibodies also do not contain

carbohydrate. A given monoclonal should be checked to verify the presence of carbohydrate

before attempting to use a periodate-mediated conjugation protocol.

Protocol

1. Dissolve the antibody to be periodate-oxidized at a concentration of 10 mg/ml in 0.01 M

sodium phosphate, 0.15 M NaCl, pH 7.2.

2. Dissolve sodium periodate in water to a ﬁ nal concentration of 0.1 M. Protect from light.

1. Preparation of Antibody–Enzyme Conjugates 803

804 20. Antibody Modiﬁ cation and Conjugation

3. Immediately add 100 l of the sodium periodate solution to each ml of the antibody solu-

tion. Mix to dissolve. Protect from light.

4. React in the dark for 15–20 minutes at room temperature.

5. Immediately quench the reaction by the addition of sodium sulﬁ te (Na

) to provide

a 2  molar excess over the initial amount of periodate added. Purify the oxidized anti-

body by gel ﬁ ltration using a desalting resin. The chromatography buffer is 0.1 M sodium

phosphate, 0.15 M NaCl, pH 7.2. To obtain efﬁ cient separation between the oxidized

antibody and excess periodate, the sample size applied to the column should be at a ratio

of no more than 5 percent sample volume to the total column volume. Collect 0.5 ml

fractions and monitor for protein at 280 nm.

6. Pool the fractions containing protein. Adjust the antibody concentration to 10 mg/ml for

the conjugation step. The oxidized antibody should be used immediately.

Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Amination

The following protocol assumes that HRP has already been periodate-oxidized by the method

of Section 1.3.

Protocol

1. Dissolve the IgG to be conjugated at a concentration of 10 mg/ml in 0.2 M sodium bicar-

bonate, pH 9.6, at room temperature. The high-pH buffer will result in very efﬁ cient

conjugation with the highest possible incorporation of enzyme molecules per antibody

molecule. To produce lower-molecular-weight conjugates, dissolve the IgG at a concen-

tration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2.

2. The periodate-oxidized enzyme (HRP) prepared in Section 1.3 was ﬁ nally puriﬁ ed using

0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. For conjugation using the lower-pH buff-

ered environment, this HRP preparation can be used directly at 10 mg/ml concentration.

For conjugation using the higher-pH carbonate buffer, dialyze the HRP solution against

0.2 M sodium carbonate, pH 9.6 for 2 hours at room temperature prior to use. A volume

of HRP solution equal to the volume of antibody solution will be required.

3. Mix the antibody solution with the enzyme solution at a ratio of 1:1 (v/v). Since an equal

mass of antibody and enzyme is present in the ﬁ nal solution, this will result in a 3.75 molar

excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-

to-antibody ratios, proportionally increase the amount of enzyme solution as required.

Typically, molar ratios of 4:1 to 15:1 (enzyme:antibody) give acceptable conjugates

useful in a variety of ELISA techniques.

4. React for 2 hours at room temperature.

5. In a fume hood, add 10 l of 5 M sodium cyanoborohydride (Sigma) per ml of reaction

solution. Caution: Cyanoborohydride is extremely toxic. All operations should be done

with care in a fume hood. Also, avoid any contact with the reagent, as the 5 M solution

is prepared in 1 N NaOH.

6. React for 30 minutes at room temperature with gentle mixing (in a fume hood).

7. Block unreacted aldehyde sites by addition of 50 l of 1 M ethanolamine, pH 9.6, per ml

of conjugation solution. Approximately a 1 M ethanolamine solution may be prepared

by addition of 300 l ethanolamine to 5 ml of deionized water. Adjust the pH of the eth-

anolamine solution by addition of concentrated HCl, keeping the solution cool on ice.

8. React for 30 minutes at room temperature.

9. Purify the conjugate from excess reactants by dialysis or gel ﬁ ltration using a desalting

resin. Use 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.0, as the buffer for either oper-

ation. The conjugate may be further puriﬁ ed by removal of unconjugated enzyme using

one of the methods described in Section 1.5, this chapter.

Conjugation of Periodate-Oxidized Antibodies with Amine or Hydrazide Derivatives

The following protocol assumes that the antibody has already been periodate-oxidized by the

method of Section 1.3 to create reactive aldehyde groups suitable for coupling with amine- or

hydrazide-containing molecules. This is an excellent method for directing the antibody modiﬁ ca-

tion reaction away from the antigen binding sites, if the antibody glycosylation points are solely

in the Fc region of the molecule. For instance, biotinylation of intact antibodies can be done

after mild periodate treatment using biotin-hydrazide (Chapter 11, Section 3) ( Figure 20.9 ).

It should be noted, however, that periodate-oxidized antibodies can self-conjugate through their

own amines if high-pH reductive amination is used. Conjugation with periodate-oxidized anti-

bodies works best if the receiving molecule is modiﬁ ed to contain hydrazide groups and the

reaction is done at more moderate pH values (e.g., slightly acidic to neutral pH).

1. For conjugation to hydrazide-containing proteins, dissolve the periodate-oxidized anti-

body at a concentration of 10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH

6.0–7.2. For conjugation to amine-containing molecules and proteins, dissolve the

oxidized antibody at 10 mg/ml in 0.2 M sodium carbonate, pH 9.6.

2. Dissolve a hydrazide-containing enzyme or other protein at a concentration of 10 mg/ml in

0.1 M sodium phosphate, 0.15 M NaCl, pH 6.0–7.2. For the preparation of a hydrazide-

activated enzyme, see Chapter 26, Section 2.4. For modiﬁ cation with a hydrazide-

containing probe, such as biotin-hydrazide, use a concentration of 5 mM in the phosphate

buffer. For conjugation through the amine groups of a secondary molecule, dissolve the

amine-containing protein at 10 mg/ml in 0.2 M sodium carbonate, pH 9.6.

3. Mix the antibody solution from step 1 with the protein solution from step 2 in amounts

necessary to obtain the desired molar ratio for conjugation. Often, the secondary molecule

is reacted in approximately a 4- to 15-fold molar excess over the amount of antibody

present.

4. React for 2 hours at room temperature.

5. In a fume hood, add 10 l of 5 M sodium cyanoborohydride (Sigma) per ml of reaction

solution. Caution: Cyanoborohydride is extremely toxic. All operations should be done

with care in a fume hood. Also, avoid any contact with the reagent, as the 5 M solu-

tion is prepared in 1 N NaOH. The addition of a reductant is necessary for stabiliza-

tion of the Schiff bases formed between an amine-containing protein and the aldehydes

on the antibody. For coupling to a hydrazide-activated protein, however, most protocols

do not include a reduction step. Even so, hydrazone linkages may be further stabilized

by cyanoborohydride reduction. The addition of a reductant during hydrazide/aldehyde

reactions also increases the efﬁ ciency and yield of the reaction.

6. React for 30 minutes at room temperature (in a fume hood).

1. Preparation of Antibody–Enzyme Conjugates 805

806 20. Antibody Modiﬁ cation and Conjugation

Figure 20.9 Polysaccharide groups on antibody molecules may be oxidized with periodate to create aldehydes.

Modiﬁ cation with biotin-hydrazide results in hydrazone linkages. The sites of modiﬁ cation using this technique

often are away from the antibody–antigen binding regions, thus preserving antibody activity.

7. Block unreacted aldehyde sites by addition of 50 l of 1 M ethanolamine, pH 9.6, per ml

of conjugation solution. Approximately a 1 M ethanolamine solution may be prepared

by addition of 300 l ethanolamine to 5 ml of deionized water. Adjust the pH of the eth-

anolamine solution by addition of concentrated HCl, while keeping the solution cool on ice.

8. React for 30 minutes at room temperature.

9. Purify the conjugate from excess reactants by dialysis or gel ﬁ ltration using desalting

resin. Use 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.0, as the buffer for either oper-

ation. The conjugate may be further puriﬁ ed by removal of unconjugated enzyme by one

of the methods of Section 1.5, this chapter.

1.4. Conjugation Using Antibody Fragments

It is often advantageous to use antibody fragments in the preparation of antibody–enzyme con-

jugates. Selected fragmentation carried out by enzymatic digestion of intact immunoglobulins

can yield lower-molecular-weight molecules still able to recognize and bind antigen. Conjugation

of these fragments with enzyme molecules can result in ELISA reagents that possess better char-

acteristics than corresponding conjugates prepared with intact antibody. Such antibody fragment

conjugates display less interference with various Fc binding proteins and also less immunogenic-

ity (due to lack of the Fc region), more facile membrane penetration for immunohistochemi-

cal staining techniques (due to lower overall conjugate molecular weight) (Wilson and Nakane,

1978; Farr and Nakane, 1981), and lower nonspeciﬁ c binding to surfaces or membranes (result-

ing in increased signal-to-noise ratios) (Hamaguchi et al., 1979; Ishikawa et al., 1981a, b).

Enzymatic digests of IgG can result in two particularly useful fragments called Fab and

F(ab )

, prepared by the action of papain and pepsin, respectively. Most speciﬁ c enzymatic

cleavages of IgG occur in relatively unfolded regions between the major domains. Papain and

pepsin, and similar enzymes including bromelain, ﬁ cin, and trypsin, cleave immunoglobulin

molecules in the hinge region of the heavy chain pairs. Depending on the location of cleav-

age, the disulﬁ de groups holding the heavy chains together may or may not remain attached to

the antigen binding fragments that result. If the disulﬁ de-bonded region does remain with the

antigen binding fragment, as in pepsin digestion, then a divalent molecule is produced [F(ab  )

]

which differs from the intact antibody by lack of an extended Fc portion. If the disulﬁ de region

is below the point of digestion, then the two heavy–light chain complexes that form the two

antigen binding sites of an antibody are cleaved and released, forming individual dimeric frag-

ments (Fab) containing one antigen binding site each (see Figure 20.4 , discussed previously).

Methods for producing immobilized papain or pepsin for antibody fragmentation can be

found in Hermanson et al. (1992). The following protocol describes the use of pepsin to cleave

IgG molecules at the C-terminal side of the inter-heavy-chain disulﬁ des in the hinge region, pro-

ducing a bivalent antigen binding fragment, F(ab  )

, with a molecular weight of about 105,000

(Figure 20.10 ). Using this enzyme, most of the Fc fragments undergo extensive degradation

and cannot be recovered intact.

Preparation of F(ab  )

Fragments Using Pepsin

1. Equilibrate by washing 0.25 ml of immobilized pepsin (Thermo Fisher) with 4  1 ml

of 20 mM sodium acetate, pH 4.5 (digestion buffer). Finally, suspend the gel in 1 ml of

digestion buffer.

2. Dissolve 1–10 mg of IgG in 1 ml digestion buffer and add it to the gel suspension.

3. Mix the reaction slurry in a shaker at 37 °C for 2–48 hours. The optimal time for com-

plete digestion varies depending on the IgG subclass and species of origin. Mouse IgG1

antibodies are usually digested within 24 hours, human antibodies are fragmented in 12

hours, whereas some minor subclasses (e.g., mouse IgG2a) require a full 48-hour diges-

tion period.

4. After the digestion is complete, add 3 ml of 10 mM Tris–HCl, pH 8.0, to the gel suspen-

sion. Separate the gel from the antibody solution using ﬁ ltration or by centrifugation.

5. Apply the fragmented IgG solution to an immobilized protein A column containing 2 ml

of gel (Thermo Fisher) that was previously equilibrated with 10 mM Tris–HCl, pH 8.0.

1. Preparation of Antibody–Enzyme Conjugates 807

808 20. Antibody Modiﬁ cation and Conjugation

6. After the sample has entered the gel, wash the column with 10 mM Tris–HCl, pH 8.0,

while collecting 2 ml fractions. The fractions may be monitored for protein by measuring

absorbance at 280 nm. The protein peak eluting unretarded from the column is F(ab  )

7. Bound Fc or Fc fragments and any undigested IgG may be eluted from the column with

0.1 M glycine, pH 2.8.

Similarly, immobilized papain may be used to generate Fab fragments from immunoglobulin

molecules. Papain is a sulfhydryl protease that is activated by the presence of a reducing agent.

Cleavage of IgG occurs above the disulﬁ des in the hinge region, creating two types of frag-

ments, two identical Fab portions and one intact Fc fragment ( Figure 20.11 ). For preparation

of the immobilized papain gel used in the following protocol, see Hermanson et al. (1992). The

gel also is commercially available from Thermo Fisher.

Preparation of Fab Fragments Using Papain

1. Wash 0.5 ml of immobilized papain (Thermo Fisher) with 4  2 ml of 20 mM sodium

phosphate, 20 mM cysteine–HCl, 10 mM EDTA, pH 6.2 (digestion buffer), and ﬁ nally

suspend the gel in 1.0 ml of digestion buffer.

2. Dissolve 10 mg of human IgG solution in 1.0 ml of digestion buffer and add it to the

immobilized papain gel suspension.

3. Mix the gel suspension in a shaker at 37 °C for 4–48 hours. Maintain the gel in suspension

during mixing. The optimal time for complete digestion varies depending on the IgG subclass

Figure 20.10 Digestion of IgG class antibodies with pepsin results in heavy chain cleavage below the disulﬁ de

groups in the hinge region. The bivalent fragments that are formed are called F(ab  )

. The remaining Fc region

normally is severely degraded into smaller peptide fragments.

and species of origin. Mouse IgG1 antibodies are usually digested within 27 hours,

whereas other mouse subclasses require only 4 hours; human antibodies are fragmented

in 4 hours (IgG1 and IgG3), 24 hours (IgG4), or 48 hours (IgG2); whereas bovine, sheep,

and horse antibodies are somewhat resistant to digestion and require a full 48 hours.

4. After the required time of digestion, add 3.0 ml 10 mM Tris–HCl buffer, pH 8.0, to the

gel suspension, mix, and then separate the digest solution from the gel by ﬁ ltration or

centrifugation at 2,000 g for 5 minutes.

5. Apply the supernatant liquid to an immobilized protein A column (2 ml gel; Thermo

Fisher) which was previously equilibrated by washing with 20 ml of 10 mM Tris–HCl

buffer, pH 8.0.

6. After the sample has entered the gel bed, wash the column with 15 ml of 10 mM Tris–HCl

buffer, pH 8.0, while 2.0 ml fractions are collected. Monitor the fractions for protein by

their absorbance at 280 nm. The protein eluted unretarded from the column is puriﬁ ed Fab.

7. Elute Fc and undigested IgG bound to the immobilized protein A column with 0.1 M

glycine–HCl buffer, pH 2.8.

Conjugation of these fragments with enzymes is done using similar methods to those previ-

ously discussed for intact antibody molecules. F(ab )

fragments may be selectively reduced in the

hinge region with DTT, TCEP, or MEA using the identical protocols outlined for whole antibody

molecules (Chapter 1, Section 4.1, and Section 1.1, this chapter). Mild reduction results in cleav-

ing the disulﬁ des holding the heavy chain pairs together at the central portion of the fragment,

thus creating two F(ab ) fragments each containing one antigen binding site ( Figure 20.12 ).

The amine groups on these fragments also may be modiﬁ ed with thiolating agents, such

as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-

activated enzymes (Section 1.1, this chapter) ( Figure 20.13 ). Amine groups further may be utilized

1. Preparation of Antibody–Enzyme Conjugates 809

Figure 20.11 Papain digestion of IgG antibodies primarily results in cleavage in the hinge region above the

interchain disulﬁ des. This produces two heavy–light chain pairs, called Fab fragments, each containing one anti-

gen binding site. The Fc region normally can be recovered intact.