Rapid hybridoma screening method for the identification of monoclonal antibodies to low-abundance cytoplasmic proteins.
Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.
O'Reilly
L A
LA
Royal Melbourne Hospital, VIC, Australia.
Cullen
L
L
Moriishi
K
K
O'Connor
L
L
Huang
D C
DC
Strasser
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology*; D008565:Membrane Proteins; D051379:Mice; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; C000606778:Bcl2l11 protein, rat; D002352:Carrier Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins