Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment.
OBJECTIVE
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.


CONCLUSIONS
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.
Li
Jun
J
Division 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
Wang
Xue-hao
XH
Wang
Xiao-ming
XM
Chen
Zhao-lai
ZL
D000081:Acetamides / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001372:Aza Compounds / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002614:Chelating Agents; D003301:Copper Radioisotopes; D005260:Female; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D011506:Proteins / Q000276:immunology*; D011877:Radionuclide Imaging
C513070:6-(p-(bromoacetamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N''' -tetraacetic acid; D000081:Acetamides; D000911:Antibodies, Monoclonal; D001372:Aza Compounds; D002614:Chelating Agents; D003301:Copper Radioisotopes; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; C549756:NBR1 protein, human; D011506:Proteins