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Updated: Aug 13, 2026
Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates
Published on: March 6, 2019
Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment
Jun Li1, Xue-hao Wang, Xiao-ming Wang
1Division of Hepatic Surgery, Department of Surgery, the First Affiliated Hospital of Nanjing Medical University and Jiangsu Province Hospital, Nanjing 210029, China. jun-li62@yeah.net
Aim:
To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 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 antigen-binding 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 site-specific 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.
