Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification.
We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.
1] Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea [2] Cancer Research Institute, Seoul National University, Seoul, Republic of Korea.
Hyori
H
Kim
Sunyoung
S
Park
Hwa Kyoung
HK
Lee
Junho
J
Chung
D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology*; D057809:HEK293 Cells; D006241:Haptens / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods; D011817:Rabbits; D057127:Single-Chain Antibodies / Q000276:immunology
D018033:Antibodies, Bispecific; D006241:Haptens; D057127:Single-Chain Antibodies