A Novel Method for Preparations of Several Hapten-Protein Conjugates

概要

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A novel method for preparations of hapten-protein conjugate was investigated using a novel coupling reagent N-(m-maleimidobenzoyloxy) succinimide (MBS) or its analogue. Haptens having amino group such as viomycin or 6-aminopenicillanic acid were introduced by a maleimide residue by the acylation with MBS under the mild aqueous condition. A hapten having hydroxyl groups but no amino group such as estradiol was introduced a maleimide residue by the reaction of the hapten and m-maleimidobenzoic acid with the presence of dicyclohexylcarbodiimide. Thus, a maleimide residue was easily introduced to haptens. The disulfide bonds of cystine residues in a protein such as bovine serum albumin (BSA) were reduced to thiol groups by the treatment of dithiothreitol. The thiol groups of the reduced BSA was coupled with the maleimide residue of the modified haptens to give hapten-protein conjugates. The yields of each conjugate was fairly good and every canjugate gave almost a single band on their sodium dodecylsulfate disk electrophoreses. Also, the introduced numbers of the hapten per molecule of BSA were easily calculated by the electrophoretic distances.