14

Conjugation of Haptens
Ranulfo Lemus and Meryl H. Karol

Abstract
Many naturally occurring proteins, peptides, carbohydrates, nucleic acids, and lipids,
as well as synthetic peptides, are successful immunogens. To elicit an immune response, a
compound must contain an antigenic determinant or epitope and must be of sufficient size
to initiate lymphocyte activation necessary for an antibody response. In practice, small
chemical compounds (haptens) are generally not good immunogens. However, when
attached to macromolecules (carriers), they can become immunogenic. An immunogen
must have epitopes that can be recognized by antigen-presenting cells and a T-cell receptor,
and it must be degradable.
Haptens and corresponding hapten–carrier conjugates have been essential to the devel-
opment of sensitive quantitative and qualitative immunoassays. In the design of hapten
conjugates, consideration must be given to the hapten, the carrier, the coupling strategy,
and the hapten density because the amount of hapten attached to the carrier influences the
strength of the immune response directed toward the newly created antigenic determinant.
Hence the haptenic density of the conjugate is also important in the development of
immunoassays. The optimal epitope density of a conjugate to elicit either a strong immune
response or provide the best immunoassay is dependent on the structure of the epitope and
the nature of the immunoassay.
The aim of this chapter is to describe the diverse techniques used to couple haptens
to carriers and provide guidance in the selection of the most appropriate procedure for a
particular hapten.

Key Words: Epitope; hapten; carrier; immunogen.

1. Introduction
Many naturally occurring proteins, peptides, carbohydrates, nucleic acids,
and lipids, as well as synthetic peptides, are successful immunogens. To elicit

From: Methods in Molecular Medicine: Allergy Methods and Protocols

Edited by: M. G. Jones and P. Lympany © Humana Press Inc., Totowa, NJ

167
168 Lemus and Karol

an immune response, a compound must contain a distinct structural component

referred to as an antigenic determinant or epitope and must be of sufficient
size to initiate lymphocyte activation necessary for an antibody response. In
practice, small chemical compounds 700–1500 Da are generally not good
immunogens. However, when attached to macromolecules, they can become
immunogenic. The small molecule is referred to as a hapten and the macro-
molecules that render haptens immunogenic are referred to as carriers. The
hapten can be thought of as an exogenous epitope attached to a carrier. The
essential features of an immunogen are (a) it must have epitopes that can be
recognized by antigen-presenting cells and a T-cell receptor and (b) it must
be degradable (1).
Haptens have diverse uses in immunology. They have been employed to
evaluate the structure of epitopes and the valence, specificity, affinity, and hetero-
geneity of antibodies. They have also been employed in antibody purification,
production of specific monoclonal antibodies, and investigation of biological
properties of antibodies. Haptens and corresponding hapten–carrier conjugates
have been essential to the development of sensitive quantitative and qualitative
immunoassays (2–4).
In the design of hapten conjugates, consideration must be given to the hapten,
the carrier, the coupling strategy, and the hapten density, i.e., the hapten:carrier
molar ratio (H/P) (5). The amount of hapten attached to the carrier influences
the strength of the immune response directed toward the newly created anti-
genic determinant (6). The haptenic density of the conjugate is also important
in the development of immunoassays. The optimal epitope density of a conju-
gate to elicit either a strong immune response or provide the best immunoassay
is dependent on the structure of the epitope and the nature of the immunoassay.
For example, several conjugates of hexamethylene diisocyanate (HDI) with
HSA were prepared in our laboratory. The molar ratio ranged from 4:1 (hap-
ten:carrier) to 49:1. The 12:1 conjugate was found to be optimum for stimulat-
ing proliferation of lymphocytes from patients with HDI hypersensitivity (7),
whereas the 36:1 conjugate was best for use in ELISA to detect IgG antibodies
(8), and a 25:1 conjugate was ideal in RAST testing to detect anti-HDI IgE
antibodies (8). Others have found a conjugate with H/P of 25:1 to yield better
binding to ELISA plates when compared with conjugates having H/P of 54:1
(9). In our experience with diisocyanate haptens, we use conjugates with H/P
of 30–40 to immunize animals (10), conjugates with H/P of 20–30 for ELISA
(11), and H/P of 10–20 for RAST.
The aim of this chapter is to describe the diverse techniques used to couple
haptens to carriers and provide guidance in the selection of the most appropri-
ate procedure for a particular hapten.
Conjugation of Haptens 169

2. Materials
2.1. Preparation of Conjugates
2.1.1. Hardware
1. Hot/stir plates.
2. Centrifuge.
3. Freeze-dryer.

2.1.2. Spontaneous Chemical Reactions

1. Hapten (isocyanates or anhydrides).
2. Carrier protein (see Table 1).
3. 0.05 M phosphate buffer (pH 7.4).
4. Monoethanolamine.
5. Whatman paper # 4.
6. 1 and 6 N HCl.
7. 0.1 M ammonium carbonate.
8. Dialysis tubing.
9. 0.15 M saline.

2.1.3. Chemicals that Require Activation

1. Hapten (bearing a carboxyl group).
2. Bovine serum albumin (BSA): 4.2 g in 220 mL 1:1 water–dioxane containing
4.2 mL of 1 M NaOH.
3. Tri-n-butylamine.
4. Dioxane.
5. Isobutylchlorocarbonate.
6. 1 M NaOH.

2.1.4. Use of Crosslinking Intermediary Molecules

2.1.4.1. CARBODIIMIDES
1. Hapten.
2. Carrier protein.
3. EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride).
4. Sodium acetate.
5. Phosphate-buffered saline (PBS) (1× conc).
6. Sodium azide.

2.1.4.2. GLUTARALDEHYDE
1. Hapten.
2. Carrier protein.
3. Glutaraldehyde.
170 Lemus and Karol

4. PBS (1× conc).

5. Sodium borohydride.
6. Sodium azide.

2.2. Characterization of Conjugates

2.2.1. Hardware
1. Spectrophotometer.

2.2.2. Determination of the Extinction Coefficient of Conjugated Haptens

(Methylene Diphenyl Diisocyanate)
1. Methylene diphenyl diisocyanate (MDI).
2. Acetone.
3. Glycine.
4. Phosphate buffer, 0.02 M (pH 7.5).

2.2.3. Spectral Analysis of Hapten Conjugates (Toluene Diisocyanate)

1. Toluene diisocyanate (TDI)–HSA conjugate.
2. Human serum albumin (HSA).
3. Phosphate buffer, 0.05 M (pH 7.4).

2.2.4. Chemical Determination of Hapten Adduction

1. Borate–KCl buffer, 0.05 M (pH 9.4). Dissolve 3.09 g boric acid and 3.73 g potas-
sium chloride in 800 mL water, adjust pH to 9.4 with NaOH, and bring to 1 L with
distilled water.
2. 0.03 M picryl sulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS).

3. Methods
3.1. Selection of a Carrier
A variety of carriers can be used for coupling with haptens. Selection of a carrier
is based on; availability of reactive sites for hapten conjugation, size, solubility
after derivatization, potential for immunogenicity, lack of in vivo toxicity, comm-
ercial availability, and cost (4,18). Proteins are most frequently used, but liposomes
(4), polysaccharides (AECM-Ficoll, dextran, agar, carboxymethyl cellulose [12–15])
and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid (16,17)
have also been employed.
Numerous proteins have been employed as carrier molecules as indicated
in Table 1. Those most frequently used include serum globulins and albumins
(6,19), keyhole limpet-hemocyanin (KLH) (20), gelatin (21), ovalbumin
(22), casein, hemocyanin (23), thyroglobulin (24), fibrinogen, and tetanus
(25), cholera (26), or diphtheria toxoid (2,27,28). The protein carrier should
be immunogenic and have a sufficient number of amino acid residues with
Conjugation of Haptens 171

Table 1
Protein Carriers Frequently Used for Conjugation with Haptens
Functional group per molecule

Protein -NH2 -SH Phenol Imidazole

carrier Mr (kDa) from Lys from Cys from Tyr from His
Keyhole 4.5 × 102  2000a 700a 1900a
Limpet 1.3 × 104 6.9b 1.7b 7.0b 8.7b
Hemocyanin
(KLH)
Human 66.5 61 1 28 16
Serum
Albumin
(HSA)
Bovine 67 59c 1 19 17
Serum
Albumin
(BSA)
Tetanus 150 106 10 81 14
Toxoid
(TT)
Ovalbumin 43 20 4 10 7
(OVA)
Adapted from S. Muller, 1999.
Abbreviations: Da, Daltons; Lys, lysine; Cys, cysteine; Tyr, tyrosine; His, histidine.
aThe amino acid composition is expressed using a molecular weight average of 5,000 kDa
(T. Hermanson, 1996).
bAmino acid groups are expressed in grams of amino acid containing this functional
group/100 g.
c30–35 of the 59 lysine residues are available for coupling.

reactive side chains for conjugation to the hapten (29). When used for production
of antibodies, conjugates can be injected into any animal except the animal of
origin for the carrier protein. For example, human, mouse, and rabbit serum
albumins have been employed for preparation of conjugates for use in guinea
pigs (31).
Haptens may be reactive or activated to a reactive state. As indicated in
Table 2, electrophilic haptens react primarily with amino and thiol moieties. The
-amino group of lysine is a good nucleophile, forming stable bonds above pH
8.0. The -NH2 group of N-terminal amino acids are less basic and are reactive
around pH 7.0. Thiols (cysteine or methionine) are the most reactive functional
group at neutral pH. Under specified conditions, haptens will react with phenols
172 Lemus and Karol

Table 2
Methods for Coupling Haptens to Proteins
Modified
Hapten amino acid Notes Reference
Direct hapten binding
Diisocyanatea -NH2, Karol, 1983
-NH2Lys,
-COOH,
SH-Cys
Acid anhydrideb -NH2, Welinder and
-NH2Lys, Nielsen, 1991
-COOH,
SH-Cys
2,4,6 Trinitrobenzene -NH2, Also used to Makela and
sulfonic acid -NH2Lys measure the free Seppala, 1986
(TNBS) amino content
of proteins
Aromatic amino acid Tyr Aromatic amino Makela and
acid converted Seppala, 1986
to diazonium
group
Carbohydrate -NH2, Coupling can Makela and
-NH2Lys occur through: Seppala, 1986
a) the reducing end,
b) carboxyl
groups of acidic
carbohydrates, or
c) via hydroxyl
groups
Cyanogen bromide -NH2, Carbohydrates Makela and
(CnBr) -NH2Lys activated by CnBr Seppala, 1986
spontaneously
couple with amino
groups
Mixed anhydride -NH2, R-COOH converted Karol and
-NH2Lys into anhydride with Tanenbaum,
isobutylchloro- 1967
carbonate
Use of coupling agents
Glutaraldehyde -NH2, Two compounds are S. Muller, 1999
-NH2Lys linked through
their amino groups
(Continued)
Conjugation of Haptens 173

Table 2 (Continued)
Modified
Hapten amino acid Notes Reference
Carbodiimides -NH2, Carboxylic groups Harlowe and
-NH2Lys are changed into Lane, 1988
reactive sites for
coupling with free
amino groups
aHDI, TDI, IPDI, MDI.
bTMA, PA.
Abbreviations: HDI: hexamethylene diisocyanate, IPDI: isophorone diisocyanate, MDI:
methylene diphenyl diisocyanate, PA: phthallic anhydride, TDI: toluene diisocyanate, TMA:
trimellitic anhydride

(tyrosine), carboxylic acids (aspartic and glutamic acids), and other amino acid
chains (arginine, histidine, and tryptophan) (30).
3.2. Preparation of Hapten Conjugates
Selection of the method for coupling hapten to protein is dictated by the
functional groups present on the hapten and the carrier, as well as the desired
orientation of the hapten for presentation to the immune system. Hapten conju-
gation to carriers can be spontaneous when the hapten is a chemically reactive
molecule such as isocyanates and anhydrides, or it can occur with the aid of a
crosslinking agent, such as glutaraldehyde or a carbodiimide, that has at least
two chemically reactive groups. Reagents frequently used to couple nonreactive
haptens to proteins are listed in Table 2.
Nonreactive haptens may be coupled to carriers using reagents that are either
incorporated into the final product or that can activate sites of the carrier protein
for subsequent linkage (Table 2). When preparing conjugates, inter- and intra-
molecular crosslinking can occur (32). Several factors that influence the extent of
crosslinking in the final conjugate are the ratio of carrier and/or hapten to coupling
agent, carrier/hapten concentration, ionic strength of the solution, temperature,
and pH (12).
3.2.1. Spontaneous Chemical Reactions
When the hapten is chemically reactive (i.e., isocyanates and acid anhydrides),
conjugation to carriers can be spontaneous reactions.
Procedure:
1. Warm 200 mL of 0.05 M phosphate buffer (pH 7.4) to 37°C in a water bath (using
a hot/stir plate), add 1 g of carrier protein (see Notes 1 and 2).
174 Lemus and Karol

2. To the middle of the rapidly stirred solution, add dropwise 110 L of hapten.
3. Stir vigorously for 1 h, then moderately for 3 h (total time 4 h).
4. Place the flask at ambient temperature, add 420 L of monoethanolamine, and stir
moderately for 1 h.
5. Filter the solution using Whatman paper #4.
6. Adjust the pH to 4.0 using 6 N HCl initially. (The starting pH is around 10.0). As
the pH nears 7, use 1 N HCl. The solution will come cloudy and protein will
precipitate (see Note 3). Cover the flask and refrigerate overnight.
7. Centrifuge solution for 20 min at 4°C at 4000 rpm.
8. Retain the supernatant until the conjugate is analyzed, then discard.
9. Dissolve the precipitate in 15 mL of 0.1 M (NH4)2CO3. Transfer the solution to
dialysis tubing and dialyze against 1 L of 0.15 M NaCl (saline) 4 h at 4°C.
10. Dialyze against distilled water for 3–4 d with 3–4 changes of distilled water/d.
11. Lyophilize the solution. Record the yield of protein.
12. Analyze the product for the amount of hapten conjugated to protein (H/P).
3.2.2. Chemicals that Require Activation – Mixed Anhydride Reaction
The method has been used for coupling steroids (33) and nucleosides (34) to
proteins.
1. Dissolve 3.05 mmol of hapten (bearing a carboxyl group) and 3.05 mmol of tri-n-
butylamine in 30 mL dioxane.
2. Cool the solution to 10°C.
3. Add 3.05 mmol of isobutylchlorocarbonate and stir at 4°C for 20 min.
4. Add mixture to a cooled solution of BSA (4.2 g in 220 mL of 1:1 water–dioxane
containing 4.2 mL of 1 M NaOH).
5. Stir the mixture for 1 h, add 2 mL NaOH, and stir for another 3 h.
6. The product should contain approximately 30 mol hapten per mol BSA (35).
3.2.3. Use of Crosslinking Intermediary Molecules
3.2.3.1. CARBODIIMIDES
Carbodiimides have the general formula R–N=C=N–R, where R and R are
aliphatic (i.e., ethylcarbodiimide) or aromatic groups (i.e., diphenylcarbodiimide).
Conjugation using carbodiimides requires the presence of and -amino and
carboxyl groups. For the most part, lysyl residues of the carrier protein and carboxyl
groups donated by the hapten are involved in the reaction. Carbodiimides also
react with sulfhydryl and phenolic residues. Although the reaction mechanism
is not fully understood, it is proposed that (1) an intermediate product (IP) is
formed that can react with an amine to give the desired peptide at low tempera-
ture by reaction between the IP and amino groups and (2) there is rearrangement
to an acyl urea, which is a side product at elevated temperatures (3,12).
The following method typically yields 50–70% conjugation of protein (see
Note 4) (1,4,12).
Conjugation of Haptens 175

1. Dissolve the hapten (up to 4 mg/mL) in 1 mL water (see Note 5).

2. Add EDC to a final concentration of 10 mg/mL with constant mixing. Adjust the
pH to 5 (see Note 6).
3. Incubate for 5 min at ambient temperature with gentle agitation.
4. Add an equal volume of carrier protein to yield a final hapten:carrier molar ratio of
20:1–40:1.
5. Stir at ambient temperature for 4 h.
6. Stop the reaction by addition of sodium acetate (pH 4.2) to a final concentration of
100 mM. Incubate at ambient temperature for 1 h.
7. Dialyze the product against 3–4 changes of PBS for 24 h to separate free hapten
from the hapten–protein conjugate (see Note 7). Store at 4°C in the presence of
sodium azide (0.02%) or in aliquots at 20°C.

3.2.3.2. GLUTARALDEHYDE
Glutaraldehyde is a bifunctional coupling reagent that can react with amine
groups to create Schiff bases or Michael-type double bond addition products. In
general, high pH favors formation of Schiff base intermediates and results in
greater conjugation, but also in higher molecular weight conjugates. Varying the
pH and the amount of glutaraldehyde added to the reaction allows some control
of the yield and the molecular weight of the conjugate. The concentration of
glutaraldehyde added generally varies from 0.2 to 2% with sporadic use of
very dilute solutions (0.05%) (4). The glutaraldehyde crosslink is very stable,
but in most cases the glutaraldehyde bridge is recognized as an epitope by the
immunized animal (1,4,12).
Glutaraldehyde crosslinking can be performed in a one- or two-step conjugation
protocol. Most frequently, the single step method is used. This method is recomm-
ended for haptens that do not contain internal lysine or cysteine residues (12).
1. Prepare a 1 mg/mL solution of the carrier protein in PBS, pH 7.4.
2. Add the hapten to the carrier protein solution using a molar ratio of 20:1–40:1
hapten:carrier protein. Cool to 4°C.
3. Just before use, prepare a 2% (v/v) glutaraldehyde solution in water.
4. Add dropwise, with constant agitation, an equal volume of 2% glutaraldehyde to
the hapten–protein carrier solution.
5. After 1 h at 4°C, stabilize the conjugate by addition of sodium borohydride (NaBH4)
to a final concentration of 10 mg/mL.
6. Incubate at 4°C for 1 h.
7. Dialyze against 3–4 changes of PBS for 24 h (see Note 7). Store at 4°C in the
presence of 0.02% sodium azide (or in aliquots at 20°C).

3.3. Use of Spacers

Hapten-directed immunogenicity may be enhanced through the use of spacers.
These molecules function to extend the distance of the hapten from the surface
176 Lemus and Karol

of the carrier. The length provided by the spacer may provide less steric hindrance
to conjugation by the hapten. The nature of the spacer may also govern the
hydrophilicity of the hapten. Generally the spacer is attached to the hapten prior
to coupling, but may also be attached to the carrier. Spacers may be immuno-
genic. With small haptens, the actual epitope will include portions of the carrier. In
this instance, the spacer will become part of the structurally known epitope, thus
reducing the size of the structurally unknown portion. Glycine and alanine residues
are the frequently used spacers (12,35).
3.4. Analysis of Conjugates
The degree of haptenation of the conjugate can be determined using radioactive
hapten tracers (3), by measurement of UV or visible light-absorbing groups on
the hapten, using non-denatured gel electrophoresis (36), amino acid analysis
(37), fluorodensitometric methods (38), gas chromatography (39), HPLC (40),
or electrospray mass spectrometry (41).
3.4.1. Spectral Analysis of Conjugates
The hapten may have an absorbance spectrum that allows it to be differen-
tiated from the carrier protein. This procedure is especially useful when the
hapten has a strong chromophore. The H/P determination by spectroscopy is
accomplished by first dialyzing the preparation to remove unbound hapten. The
absorption of the conjugate is measured at the max for the hapten, then the
protein concentration of the conjugate is determined from absorbance at
280 m. The result is corrected for the A280 nm because of the hapten. The H/P
is calculated using Beer’s law: A = Cl, where  is the extinction coefficient,
A the molar absorbance, C the molar concentration, and l the path length (6,30).
It should be noted that inaccuracy might result if the spectral characteristics
of the hapten change when conjugated to the protein (30). The following
assumptions are made when hapten conjugates are analyzed by UV absorbance:
(1) the solution is free of noncovalently bound hapten, (2) conjugates are soluble
in the matrix used for analysis, and (3) chromophores of the carrier protein and
hapten do not change on conjugation (6).
3.4.1.1. DETERMINATION OF THE EXTINCTION COEFFICIENT OF CONJUGATED MDI
The extinction coefficient of the free hapten and protein can be obtained
from tables or handbooks. That of the conjugated form can be calculated using
the following approach (32):
1. MDI (Mondur M, Bayer USA, Pittsburgh, PA) was recrystallized, then dissolved
in acetone at 2.1 mg/mL.
2. A total of 10 L of the solution was added to 0.16–2.8 mM glycine in 0.02 M
phosphate buffer, pH 7.5.
Conjugation of Haptens 177

Table 3
Determination of the Extinction Coefficient of MDI Following Carbamoylation
of the Nucleophile
Molar
Absorbance at extinction
Glycine (mg) MDI (g) max (nm) max coefficient
0.2 21 245.1 0.415 24714
2.0 21 246.8 0.480 28577
20 21 248.8 0.592 35238
200 21 248.8 0.615 36613
Adapted from Jin and Karol, 1988.

3. The absorbances of the solutions were determined at 200–350 nm. See Table 3.
4. The extinction coefficients were calculated from Beer’s law: A = Cl (see
Section 3.4.1.).

3.4.1.2. SPECTRAL ANALYSIS OF TDI CONJUGATES

When there is overlap in the spectra of the hapten and the carrier, H/P can
be determined from a “difference” spectrum (see Fig. 1). The hapten can be
quantified by subtracting the absorbance of the uncoupled protein carrier at the
haptenic max from that of the conjugate (42,43).
1. Separately dissolve 3 mg of the TDI–HSA conjugate and 3 mg of HSA in 0.05 M
of phosphate buffer, pH 7.4 to a final concentration of 600 g/mL.
2. Determine the absorbance from 600 to 190 nm of the following solutions:
a. carrier protein vs buffer,
b. conjugate vs buffer,
c. conjugate vs carrier protein.
3. Calculate H/P using Beer’s law with the absorbance value of the conjugate obtained
from 2c.

3.4.2. Chemical Determination

When the hapten does not have absorbance in the UV or visible range, the
extent of hapten coupling to protein can be estimated from the determination of the
number of remaining amino groups on the carrier protein. 2,4,6-Trinitrobenzene
sulfonic acid (TNBS) is frequently used for this estimation. Haptenic content of
conjugates is determined from absorption of the trinitrophenyl derivative of the
protein before and after conjugation (3,37).
1. Separately prepare 1% solutions of the carrier protein and conjugate in 0.05 M
borate–KCl buffer, pH 9.4.
178 Lemus and Karol

Fig. 1. Ultraviolet absorption spectra. Left: Ovalbumin (OVA) and p-tolylureido-

ovalbumin (T-OVA) conjugate each 1 mg/ml, 0.05 M phosphate buffer, pH 7.0. Right:
the difference spectrum of T-OVA versus OVA (T-OVA vs. OVA).

2. Prepare assay tubes according to Table 4.

3. Prepare 0.03 M picryl-sulfonic acid (TNBS; Sigma, Cat # p-2297) in distilled water
using a glass beaker.
4. Add 50 L of TNBS to each tube.
5. Vortex, let sit at ambient temperature for 30 min.
6. Immediately thereafter, read absorbance 420 nm (do not use quartz cuvettes) (see
Note 8).
Conjugation of Haptens 179

Table 4
Set Up for Conjugate Analysis by TNBS
Volume of borate Volume of conjugate Volume of protein
buffer (ml) solution (l) solution (l)
2* — —
2* — —
1.95 50 (500 g) —
1.90 100 (1 mg) —
1.85 150 (1.5 mg) —
1.95 — 50 (500 g)
1.90 — 100 (1 mg)
1.85 — 150 (1.5 mg)
* Blanks.

7. Read blanks vs H2O, then samples against the lowest blank.

8. Calculate the hapten substitution using the following formula:
[100(Absorbance conjugate)]
9. %Substitution =100 
(Absorbance carrier protein)

4. Notes
1. In general, conjugates of serum albumin are more soluble than those of globulins
or ovalbumin. Because of its large size, KLH is more likely to precipitate during
crosslinking conjugation. To preserve the solubility and stability of the conjugate,
coupling reactions using KLH should be performed under conditions of high salt
(at least 0.9 M NaCl) (4).
2. The haptenic content (H/P) of the final conjugate will vary with (1) the amount of
hapten added, (2) pH of the solution with higher pH usually favoring greater substitu-
tion, (3) incubation temperature (higher temperatures favor higher substitution).
3. Conjugates having low hapten substitution will usually not precipitate at pH 4.
Adjust the pH to 7 before starting dialysis.
4. Adding 5 mM N-hydroxysulfosuccinimide may enhance the efficiency (12).
5. To avoid amino acid modification in the hapten, you may block it with citraconic
anhydride (CA). For this, dissolve the hapten in water to 10 mg/mL. Adjust the pH
to 8.5 and stir at room temperature. Prepare a 10 mg/mL solution of CA in water
and slowly add an equal volume to the hapten solution and incubate for 1 h.
Following the reaction with ECD, the citraconylated amino groups are depro-
tected by dialysis against 5% acetic acid. Finally, dialyze the conjugate against
PBS (1,12).
6. Adding N-methyl-imidazole to the coupling mixture may decrease the formation of
acylurea adducts (12).
180 Lemus and Karol

7. Larger haptens may not dialyze efficiently. Gel filtration can be used to separate the
free hapten from the conjugate.
8. If the absorbance of the conjugate is equal to that of the carrier protein, H/P is very
low. A more sensitive method of analysis is needed (such as electrospray mass
spectrometry).

Acknowledgment
This work was supported by grant # ES 05651 from the National Institute of
Environmental Health Sciences, USA.

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