用于免疫酶量测定的单体2,4-二硝基苯基Fab'-β-D-半乳糖苷酶缀合物的制备。

Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay.

作者信息

Imagawa M, Hashida S, Ishikawa E, Freytag J W

出版信息

J Biochem. 1984 Dec;96(6):1727-35. doi: 10.1093/oxfordjournals.jbchem.a135005.

DOI:10.1093/oxfordjournals.jbchem.a135005

Abstract

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.

摘要

本文描述了一种制备单体Fab'-β-D-半乳糖苷酶缀合物的方法，这是开发灵敏免疫酶测定法所必需的。抗人IgG F(ab')2用2,4-二硝基苯基基团标记，通过还原分裂成Fab'，并与使用N,N'-邻苯二甲酰亚胺通过硫醇基团引入β-D-半乳糖苷酶的过量马来酰亚胺基团反应。随后，通过在(抗2,4-二硝基苯基)IgG-琼脂糖4B柱上进行亲和色谱，将单体2,4-二硝基苯基Fab'-β-D-半乳糖苷酶缀合物与未缀合的β-D-半乳糖苷酶分离。在单体缀合物制备中，98%的β-D-半乳糖苷酶活性与Fab'相关，90%与特异性(抗人IgG)Fab'相关。这种缀合物允许通过免疫酶测定技术测量0.1 fmol的人IgG。