Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.
This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.
Andersen
Ditte C
DC
Department of Immunology and Microbiology, University of Southern Denmark, Odense C, Denmark.
Jensen
Charlotte H
CH
Gregersen
Annemette
A
Brandt
Jette
J
Kliem
Anette
A
Skjødt
Karsten
K
Koch
Claus
C
Teisner
Børge
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000276:immunology*; D001710:Biotin / Q000737:chemistry*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000941:Antigens; D017077:Culture Media, Conditioned; D000939:Epitopes; D006023:Glycoproteins; D001710:Biotin