Enzyme Immunoassay of Blasticidin S with High Sensitivity: A New and Convenient Method for Preparation of Immunogenic (Hapten-Protein) Conjugates

An antibody against blasticidin S (BLS), an antibiotic effective for blast disease of rice, has been produced in rabbits immunized with a blasticidin S-protein conjugate prepared by a novel and convenient procedure devised to couple BLS to bovine serum albumin (BSA) after sodium borohydride reduction of its disulfide bonds, using N-(m-maleimidobenzoyloxy)succinimide (MBS) as a cross-linker. BLS-MBS-BSA conjugate contained about 16 BLS per BSA molecule. Enzyme labeling of BLS with β-d-galactosidase was performed by utilizing another cross-linker, N-(γ-maleimidobutyryloxy)succinimide by means of a convenient labeling method which we introduced last year. A double antibody enzyme immunoassay of BLS which could determine as little as 100 pg per tube of BLS was developed using labeled BLS and anti-BLS antiserum. Various commonly used drugs were found to have little reactivity in this immunoassay, indicating that the anti-BLS produced is highly specific. The titer of the anti-BLS was excellent and 10,000,000-fold diluted solution could bind with the enzyme labeled BLS.