Coupling Fab′ fragment of rabbit anti‐human IgG antibody to β‐d‐galactosidase and a highly sensitive immunoassay of human IgG

Enzyme immunoassays with special reference to ELISA techniques.

“…Kato et al (1975aKato et al ( , 1976a used the double antibody sandwich method, which together with a fluorogenic substrate resulted in an exceedingly sensitive assay.…”

Sandwich Enzymoimmunoassay of Hepatitis B Surface Antigen (HBsAg)

“…The antiserum was absorbed three times with 1/20vol of human serum which was shown to be free of HBsAg by RIA. After the absorption an IgG fraction was prepared by fractionation with (NH,),SO, [6] followed by passage through a column of Adachi/Fukuda/Funahashi/Kurahori/Ishi kawa DEAE-cellulose [8]. Purified anti-HBsAg was obtained by affinity chromatography.…”

“…The IgG fraction of anti-HBsAg, which was not purified by affinity chromatography, was digested with pepsin followed by reduction with 2-mercaptoethylamine to obtain Fab' fragments as described previously [6]. The Fab' fragments were treated with excess N,N-o-phenylenedimaleimide (Aldrich Chemicals Company, Inc., Milwaukee) and the maleimide residues introduced into the Fab' fragments were allowed to react with sulfhydryl groups of B-D-galactosidase from E. coli (Boehringer, Mannheim) [5].…”

Monoconjugate enzyme linked immunoassay

“…The periodate method [2] yields heterogeneous conjugates [3]. The dimaleimide method [4] produces conjugates of limited heterogeneity which are likely to be unstable [5] ; these conjugates have however yielded the most sensitive enzyme immunoassays so far reported [6].…”