Preparation of Monoclonal Antibodies to the Avian Progesterone Receptor*

Abstract

In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated alpha PR 6, 11, 13, 16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradients, and in recognizing receptor proteins that were resolved by denaturing gel electrophoresis and transferred to nitrocellulose (Western blots). Immunoisolation of receptor was also demonstrated using receptor labeled covalently with the synthetic progestin, R5020. The antibodies were specific for progesterone receptor and did not cross-react with estrogen receptor from the oviduct or glucocorticoid receptor from chick liver. Two antibodies, alpha PR 6 and alpha PR 22, also recognized some mammalian forms of the progesterone receptor. Both antibodies reacted with progesterone receptor from the rabbit uterus and alpha PR 6 recognized human progesterone receptor. Four of the antibodies recognized both A and B forms of the avian receptor while alpha PR6 was specific for the B form.