Rapid hybridoma screening method for the identification of monoclonal antibodies to low-abundance cytoplasmic proteins
- Author(s)
- O'Reilly, LA; Cullen, L; Moriishi, K; O'Connor, L; Huang, DCS; Strasser, A;
- Details
- Publication Year 1998-11,Volume 25,Issue #5,Page 824-+
- Journal Title
- BIOTECHNIQUES
- Publication Type
- Journal Article
- Abstract
- 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 all 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 nse (GST)-Bim(L) 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, nod most monoclonal antibodies identified were proven to De useful for immunofluorescence staining and other applications.
- Publisher
- EATON PUBLISHING CO
- Keywords
- CELL-LINES; BCL-2; ANTIGENS; ASSAY; MOUSE; IMMUNOGLOBULIN; PURIFICATION; EXPRESSION; DISTINCT; GROWTH
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 1998-11-01 12:00:00