Kitagawa T, Fujitake T, Taniyama H, Aikawa T
J Biochem. 1978 May;83(5):1493-501. doi: 10.1093/oxfordjournals.jbchem.a132059.
A new cross-linking reagent of the hetero-bisfunctional type, a N-(maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of beta-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4 ng of the antibiotic could be detected.
制备了一种新型的异双功能交联剂N-(马来酰亚胺苯甲酰氧基)琥珀酰亚胺(MBS)，并在温和的水相条件下通过两步法将其用于紫霉素的酶标记。第一步，用有限量的MBS将马来酰亚胺残基选择性地引入紫霉素的N1-氨基上。第二步是在马来酰亚胺残基与β-D-半乳糖苷酶的游离巯基之间形成硫醚。用紫霉素-BSA偶联物免疫家兔制备了抗紫霉素血清。通过用乙酰基保护紫霉素的N6-氨基，使N1-氨基琥珀酰化，用混合酸酐法活化羧基并将其与牛血清白蛋白(BSA)的氨基偶联来制备偶联物。基于紫霉素及其酶偶联物与抗血清稀释液之间的竞争，通过酶免疫测定法证明了抗血清的特异性。通过使用紫霉素-酶偶联物和抗紫霉素血清，采用双抗体法通过竞争结合程序成功地进行了紫霉素的酶免疫测定，可检测到0.1至4 ng的抗生素。
