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

Journal Article · Tue Sep 22 04:00:00 EDT 1987 · Biochemistry; (United States)

OSTI ID:5400320

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

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.

• Research Organization: Univ. of Colorado Health Sciences Center, Denver
• OSTI ID: 5400320
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:19; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMAL CELLS
ANIMALS
ANTIBODIES
BODY
ELECTROPHORESIS
GLANDS
HORMONES
IMMUNOLOGY
ISOTOPE APPLICATIONS
KETONES
LABELLED COMPOUNDS
MAMMALS
MAMMARY GLANDS
MAN
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MONOCLONAL ANTIBODIES
ORGANIC COMPOUNDS
ORGANS
PREGNANES
PRIMATES
PROGESTERONE
PROTEINS
RADIORECEPTOR ASSAY
RECEPTORS
STEROID HORMONES
STEROIDS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
VERTEBRATES