Determination of the epitope specificities of monoclonal antibodies using unprocessed supernatants of hybridoma cultures.
A double monoclonal antibody (mAb) ELISA has been developed to determine the epitope specificities of murine monoclonal (mAbs). It permits the mAbs which bind to the same or adjacent epitopes to be distinguished from those which bind to separate epitopes on soluble monomeric proteins. The assay is designed for the early evaluation of mAbs in hybridoma culture supernatants when purified or labeled mAbs are not available. It does not rely on chemical or enzymatic fragmentation of the antigen and does not generate results which may be due to differences in the affinities of the mAbs. Moreover the characteristic multiple binding of polyclonal antibodies to the same epitope is also avoided. A further advantage is the accessibility of epitopes on a given antigen since the antigen is presented by a solid-phase mAb, in contrast to assays in which the antigen itself is adsorbed onto the solid phase. The test was evaluated using culture supernatants from hybridomas which produced mAbs against the complement proteins C3a, C6 or C7.
Department of Immunology, Georg-August-University, Göttingen, F.R.G.
R
R
Würzner
M
M
Oppermann
R
R
Zierz
H
H
Baumgarten
O
O
Götze
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001710:Biotin; D015926:Complement C3a / Q000276:immunology; D003183:Complement C6 / Q000276:immunology; D003184:Complement C7 / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D005069:Evaluation Studies as Topic; D006825:Hybridomas / Q000276:immunology*; D007158:Immunologic Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D013154:Spleen / Q000276:immunology
D000911:Antibodies, Monoclonal; D003183:Complement C6; D003184:Complement C7; D000939:Epitopes; D001710:Biotin; D015926:Complement C3a