Polyclonal Antibodies to Oestrogen Receptors of Human Myometrium

Rabbit antibodies to oestrogen receptor of human origin were utilized for investigating oestrogen receptor distribution, quantification and translocation in oestradiol target cells. The purification of oestrogen receptor to homogeneity has been achieved. A single protein band was obtained at 65,000 molecular weight under denaturing conditions. The purified oestrogen receptor also had a sedimentation value in the region of 4S. The antibodies to the affinity purified oestrogen receptor had been produced in rabbit and a significant titre was found 3 months after primary immunization. The antibodies obtained had been shown to contain specific antibodies to the receptor protein by four criteria: 1. precipitation of the radioactive steroid after the addition of goat anti-rabbit antibody to a mixture of the [3H] oestradiol-receptor complex and the immunoglobulin. Using the double antibody precipitation technique, the titre of the antibodies was also determined, 2. adsorption of the oestradiol-receptor complex by immunoglobulin linked to Sepharose, 3. the ability of the immunoglobulin to increase the sedimentation coefficient of the oestradiol-receptor complex. Using [3H] oestradiol as the marker for oestradiol-receptor complex, it is possible to observe shifts in the sedimentation coefficients of oestrogen receptor in the absence and the presence of the antibody under both low and high salt conditions, 4. the demonstration of translocation of receptor in ZR-75-1 cells. The immunofluorescent method, using anti-oestrogen receptor antibodies as primary and goat anti-rabbit fluorescein isothiocya-nate conjugated as secondary antibody, was employed for immuno-histochemical localization of oestrogen receptor. The frozen sections from rat and human uterine tissues, oestrogen receptor positive human breast cancer and the ZR-75-1 cell line all showed cross-reaction. No immunoreaction was observed in tissues from rat spleen and oestrogen negative human breast tissues. In vitro translocation sudies were also carried out on ZR-75-1 cells and human myometrial tissues when they were incubated with diethyl-stiboestrol at 3