Monoclonal antibodies recognizing the nuclear binding sites of the avian oviduct progesterone receptor.
Monoclonal antibodies which block the binding of the avian oviduct progesterone receptor (PR) to nuclear acceptor sites have been prepared. We have previously shown that such acceptor sites consist of complexes of specific nonhistone proteins and DNA. The antigen was a reconstituted, enriched nuclear site for avian oviduct PR formed by reannealing a partially purified acceptor protein to pure hen DNA to reconstitute native-like acceptor sites. These reconstituted acceptor sites were then partially digested with deoxyribonuclease I and injected into BALB/c mice. The spleen cells were fused with NS-1 mouse myeloma cells. Hybridomas were then grown and tested for the ability of their culture media to 1) inhibit PR binding to avian oviduct nucleoacidic protein (NAP) which contains the native-like acceptor sites, but 2) to not inhibit PR binding to pure hen DNA. Three hybridoma clones produced ascites fluids which inhibited PR binding to intact oviduct chromatin and NAP but not to pure hen DNA. Control ascites fluids, prepared against other protein antigens, showed no inhibition of PR binding. The three positive ascites fluids contained low concentrations of immunoglobulin (0.3-0.5 mg/ml). The antibodies did not affect the stability of the receptor and did not interact with PR when analyzed by sucrose density gradient sedimentation. Direct binding of the antibodies to the NAP is shown by an enzyme-linked immunosorbent assay. The monoclonal antibodies display a partial species specificity with regard to the source of NAP in that PR binding to hen oviduct NAP was inhibited by greater than 90%, while PR binding to human uterine NAP was inhibited less than 40% by the antibodies. Further characterization of these candidate antiacceptor site monoclonal antibodies with regard to tissue, species, and steroid receptor specificities are underway.
Goldberger
A
A
Littlefield
B A
BA
Katzmann
J
J
Spelsberg
T C
TC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D001202:Ascitic Fluid / Q000276:immunology; D002467:Cell Nucleus / Q000276:immunology*; D002645:Chickens; D002843:Chromatin / Q000276:immunology; D004247:DNA / Q000276:immunology; D005260:Female; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D009698:Nucleoproteins / Q000276:immunology; D010057:Oviducts / Q000276:immunology*; D011388:Prolactin / Q000378:metabolism; D011980:Receptors, Progesterone / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D002843:Chromatin; D009698:Nucleoproteins; D011980:Receptors, Progesterone; D011388:Prolactin; D004247:DNA