enzyme-linked immunosorbent assay (original) (raw)

2016

Abstract

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver gluco- corticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR- antibodies by the hybridomas was carried out with an enzyme- linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using (3H)triamcinolone aceto- nide-GR complex from rat liver cytosol as tracer. Hybrid- omas from 10 different microplate wells, positive in both as- says, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal anti- bodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hy- bridoma cell lines secreted IgM antibodies; two, IgGl; three, IgG2a; and one, IgG2b. The GR-antibody complex was iden- tified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with mono- clonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactiv- ity to the mouse liver GR varied among the monoclonal anti- bodies. No cross-reactivity was observed to the human lym- phocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the

Sam Okret hasn't uploaded this paper.

Let Sam know you want this paper to be uploaded.

Ask for this paper to be uploaded.