Abstract
A novel method for enzyme immunoassay of penicillin has been developped. The specific antiserum to penicillin was obtained by a new method. Disulfide bonds of cystine residues in bovine serum albumine (BSA) was reduced to thiol groups with dithiothreitol. An amino group of ampicillin was coupled with N-(m-maleimidobenzoyloxy) succinimide (MBS) and the resulting MBS-acylated ampicillin was coupled with the reduced thiol groups of BSA. Injections of ampicillin-BSA conjugate, emulsified with complete Freund's adjuvant to a rabbit yielded anti-penicillin serum. A new cross linking reagent N-(3-maleimidopropionylglycyloxy) succinimide was prepared and used for enzyme labeling of ampicillin. The labeling with β-galactosidase was performed in aqueous mild conditions by two stage reactions by the similar method reported previously for enzyme labeling of insulin and viomycin with MBS. The enzyme labeled ampicillin was purified by a chromatography on a Sepharose 6B column and the peak fraction in the enzyme activity was used for enzyme immunoassay. Enzyme immunoassay of penicillin was performed by the competitive binding procedure with the double antibody method. Sensitivity of the assay was 40μg/litter. Cross reactivity of penicillin G and cephaloglycin was tested and the assay method clearly distinguished cephalosporin from penicillin.
