An automatic fluorescence micro-ELISA system for quantitative screening of hybridoma supernatants using a protein-A-beta-galactosidase conjugate.
We report on a rapid micro-ELISA screening procedure for the detection of monoclonal antibodies directed against cell surface determinants on lymphoid cells of the mouse. This method employs Terasaki-type trays coated with a monolayer of target cells fixed with 0.02% glutaraldehyde. Cells are incubated with monoclonal antibodies, followed by affinity column-purified rabbit-anti-rat immunoglobulin antibodies and a protein-A-beta-galactosidase conjugate. Binding of antibodies to the cells is visualized by incubation with the substrate 4-methylumbelliferyl galactoside. Fluorescence in the individual wells of the Terasaki trays is then quantitatively analysed within 120 s using a scanning inverted microfluorometer, connected to a digital voltmeter and a desk-top calculator.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005696:Galactosidases; D006825:Hybridomas / Q000276:immunology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008832:Microchemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013205:Staphylococcal Protein A; D013601:T-Lymphocytes / Q000032:analysis; D001616:beta-Galactosidase
D000911:Antibodies, Monoclonal; D013205:Staphylococcal Protein A; D005696:Galactosidases; D001616:beta-Galactosidase