Hashida S, Imagawa M, Ishikawa E, Freytag J W
J Immunoassay. 1985;6(1-2):111-23. doi: 10.1080/01971528508063024.
A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods. The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.
描述了一种将亲和纯化的Fab'与来自大肠杆菌的辣根过氧化物酶和β-D-半乳糖苷酶偶联的简单方法。IgG经过胃蛋白酶消化、用2-巯基乙胺还原、亲和纯化以及与引入酶中的马来酰亚胺基团反应等连续过程。在本方法中，在最后一步仅需进行一次凝胶过滤以从未偶联的组分中分离偶联物，而在先前方法中凝胶过滤必须重复3-4次。通过本方法获得的偶联物制剂中非特异性偶联物较少，并且与通过先前方法获得的制剂相比，免疫酶测定技术产生的背景较低。
