Hapten—carrier conjugation hydrogens

Active-Hydrogen-Mediated Hapten-Carrier Conjugation... 

Hermanson G. T. and Smith R. K., Use of the Munnich reaction for immobilization or conjugation of active hydrogen containing compounds-preparation of affinity supports and hapten carrier conjugates, EASES J., 6(5), A1729-A1729, 1992. 

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. 

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. 

Maleimides (Section 11.2.3.2.1.2) are particularly useful for the conjugation of thiol-containing haptens. Other approaches are based on the activation of the protein with bromoacetamide groups (Singh et al., 1978) or on the formation of disulfide bonds between the carrier and hapten in acetate buffer, pH 4.0, in the presence of hydrogen peroxide, as used for the conjugation of penicillenic acid (De Week and Eisen, 1960). 

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