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Title: Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies

Abstract

A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors cross-reacts with the M/sub r/ 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with (/sup 3/H)promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. B receptors purified under these conditions are transformed and biologically active. They were maintained as undergraded 120-kDa doublets and retained both hormone and DNA binding activities. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG/sub 1/), B-64 (IgG/sub 1/), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG/sub 1/), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displacemore » the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Colorado Health Sciences Center, Denver
OSTI Identifier:
5400320
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 26:19
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; PROGESTERONE; RADIORECEPTOR ASSAY; RECEPTORS; IMMUNOLOGY; MOLECULAR STRUCTURE; ELECTROPHORESIS; MAMMARY GLANDS; MAN; MONOCLONAL ANTIBODIES; TRITIUM COMPOUNDS; TUMOR CELLS; ANIMAL CELLS; ANIMALS; ANTIBODIES; BODY; GLANDS; HORMONES; ISOTOPE APPLICATIONS; KETONES; LABELLED COMPOUNDS; MAMMALS; MEMBRANE PROTEINS; ORGANIC COMPOUNDS; ORGANS; PREGNANES; PRIMATES; PROTEINS; STEROID HORMONES; STEROIDS; TRACER TECHNIQUES; VERTEBRATES; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Estes, P.A., Suba, E.J., Lawler-Heavner, J., Elashry-Stowers, D., Wei, L.L., Toft, D.O., Sullivan, W.P., Horwitz, K.B., and Edwards, D.P. Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies. United States: N. p., 1987. Web.
Estes, P.A., Suba, E.J., Lawler-Heavner, J., Elashry-Stowers, D., Wei, L.L., Toft, D.O., Sullivan, W.P., Horwitz, K.B., & Edwards, D.P. Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies. United States.
Estes, P.A., Suba, E.J., Lawler-Heavner, J., Elashry-Stowers, D., Wei, L.L., Toft, D.O., Sullivan, W.P., Horwitz, K.B., and Edwards, D.P. Tue . "Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies". United States.
@article{osti_5400320,
title = {Immunologic analysis of human breast cancer progesterone receptors. 1. Immunonaffinity purification of transformed receptors and production of monoclonal antibodies},
author = {Estes, P.A. and Suba, E.J. and Lawler-Heavner, J. and Elashry-Stowers, D. and Wei, L.L. and Toft, D.O. and Sullivan, W.P. and Horwitz, K.B. and Edwards, D.P.},
abstractNote = {A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors cross-reacts with the M/sub r/ 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with (/sup 3/H)promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. B receptors purified under these conditions are transformed and biologically active. They were maintained as undergraded 120-kDa doublets and retained both hormone and DNA binding activities. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG/sub 1/), B-64 (IgG/sub 1/), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG/sub 1/), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 26:19,
place = {United States},
year = {Tue Sep 22 00:00:00 EDT 1987},
month = {Tue Sep 22 00:00:00 EDT 1987}
}