Haptens conjugation

The Mannich reaction can be used for the immobilization of certain drugs, steroidal compounds, dyes, or other organic molecules that do not possess the typical nucleophilic groups able to participate in traditional coupling reactions (Hermanson et al., 1992). It also can be used to conjugate hapten molecules to carrier proteins when the hapten contains no convenient nucleophile for conjugation (Chapter 19, Section 6.2). In this case, the carrier protein contains the primary amines and the hapten contains at least one sufficiently active hydrogen to participate in the condensation reaction. 

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. 

In optimizing an assay during development the nature of the interaction of the analyte with the antibody is particularly important. Assays usually are carried out under physiological conditions and frequently no effort is made to optimize for pH, ionic strength, or other factors. These factors can directly affect the assay by modifying the presentation of the soluble analyte to the antibody or changing the interaction of the antibody and the conjugated hapten used in the assay. For example, assays for some compounds show a distinct pH dependence. In an indirect competitive... 

The production of haptens for conjugation is the last point in the assay development process where rigorous structural analysis is possible. Because the assay specificity is determined by the structure(s) of the conjugated haptens, it is critically important for these compounds to be pure and structurally well characterized before conjugation renders this impossible. 

Microtiter plates were coated with 1 or 10 p ell of hapten-chicken egg ovalbumin conjugates (hapten-OVA). The retronamine coryugate provided a more sensitive assay with a more concentrated coating solution (10 g/well) than the retronecine conjugate, which was used at a 1 pg/well concentration. Both concentrations were tested with each assay. The concentration which provided the more sensitive assay was selected for later experiments. Standard concentrations of the hapten were assayed on each microtiter plate. The standards (reoonecine and... 

Gabor, R, Hamilton, G. and Pittnei F. (1995) Drug-protein conjugates Haptenation of 1-methyl-1 Oa-methoxydihydrolysergol and 5-bromonicotinic acid to albumin for the production of epitope-specific monoclonal antibodies against nicergoline. J. Pharm. Sci., 84,1120-1125. 

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