Open AccessSite‐specific conjugation of bifunctional chelator BAT to mouse IgG 1 Fab 1 fragment
Author(s) -
LI Jun,
WANG Xuehao,
WANG Xiaoming,
CHEN Zhaolai
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00242.x
Subject(s) - conjugate , chemistry , bifunctional , cysteine , thiol , immunoglobulin fab fragments , chelation , maleimide , monoclonal antibody , stereochemistry , antibody , enzyme , biochemistry , peptide sequence , biology , catalysis , organic chemistry , immunology , mathematical analysis , mathematics , gene , complementarity determining region
Aim: To perform a site‐specific conjugation of Fab′ fragments of a mouse monoclonal antibody(MoAb) B43(of IgG 1 subtype) to a bifunctional chelator 6‐[ p ‐ (bromoacetamido) benzyl]‐1,4,8,11‐tetraazacyclotetradecane‐ N,N′,N″,N′″ ‐tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab′. Methods: B43 was cleaved using a simple 2‐step method. First, stable F(ab′) 2 was produced by pepsin treatment. Fab′ with free thiol in the hinge region was then obtained by cysteine reduction of F(ab′) 2 . Second, a sitespecific conjugation of Fab′ to thiol‐specific BAT was performed in a one‐step reaction. Results: The Fab′ fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab′ and 74% of the initial concentration of Fab′, respectively. The F(ab′) 2 , Fab′ and Fab′‐BAT all maintained reasonable antigen‐binding properties. 67 Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab′‐BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab′‐ BAT, which can be used to make radiometal‐labeled conjugates for further diagnostic and therapeutic applications.