Hapten coupling, methods

Labeling of Antigens and Antibodies with Enzyme. Many methods exist for coupling haptens, proteins, and carbohydrates to proteins. Some of the most commonly used are summarized in the table together with some selected references. An extensive review on protein-protein coupling reactions has recently appeared and could be used as a source of additional chemical details on the coupling methods. 

A method similar in concept was described by Burd et al. (1977b) using haptens coupled via an ester bond to umbelliferone. Hydrolysis of the ester by porcine liver esterase is followed by the liberation of the fluorescent umbelliferone. This hydrolysis, however, is prevented or sharply reduced by the presence of antibody to the hapten free hapten present in the test sample competes for the available antibody and more hydrolysis is produced by the enzyme. 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

Cohen et al., 2007), and an improved method for coupling synthetic peptide haptens to carrier proteins (Lateef et al., 2007). 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

The crosslinking scheme using this method can make use of the native e- and N-terminal amines on carrier proteins as the source of primary amine for the condensation reaction. Added to the conjugation reaction then is formaldehyde and the desired hapten to be coupled containing an appropriately active hydrogen. 

To increase the yield of conjugated hapten using this procedure, cBSA is used as the carrier protein in the method described below (see Section 2.1, this chapter for additional information on this carrier). The greater density of amine groups on cBSA available for participation in the Mannich reaction over that available on native proteins provides better results in coupling active-hydrogen-containing haptens. 

Hosoda H, Ishikawa E (1993) Coupling of haptens to carriers and to labels. In Masseyeff RF, Albert WH, Staine NA (eds.) Methods of immunological analysis. Samples and reagents. VCH, Weinheim, p... 

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