Production of monoclonal antibodies to human estrogen‐receptor protein (ER) using recombinant ER (RER)

Abstract

Two monoclonal antibodies (MAbs), IC5 and ID5, were produced using spleen cells from BALB/c mice immunized with recombinant estrogen-receptor protein (RER). On immunoblotting, both MAbs reacted with the 67-kDa polypeptide chain obtained by transformation of E. coli and transfection of COS cells with plasmid vectors expressing ER. The epitopes of both MAbs were in the N-terminal domain (A/B region) of the receptor. In normal human tissues, IC5 and ID5 reacted with cells known to contain large amount of ER, such as cells of the mammary gland and the uterus. Staining was localized predominantly in nuclei with little or no cytoplasmic reactivity. IC5 and ID5 were unreactive with tissues usually considered to be negative for ER. The reactions of these 2 MAbs were further tested on different tumor types, using immunohistochemical (IHC) method on frozen sections. In breast cancer, a good correlation was found between the results obtained on frozen sections and those using the conventional radioligand dextran-coated charcoal (DCC) assay. Immunostaining with IC5 and ID5 MAbs was also assessed on routinely processed paraffin sections using the antigen-retrieval method. Staining was comparable to that obtained on frozen sections in virtually all the breast carcinomas. Negative reactions were consistently obtained with both antibodies on human neoplasms derived from other non-estrogen-dependent organs. IC5 and ID5 MAbs may thus be of value in routine diagnostic histopathology for assessment of the estrogen-receptor content in human carcinomas.

Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice

“…Second, the interpretation of the staining at the cellular level is easier in paraffin wax sections compared with autoradiograms and immunohistochemically stained frozen sections. Finally, the commercially available ER-antibody used, which has been documented as ER-α-specific by Saati et al (1993) and Pettersson et al (1997) has made it possible to immunodetect ER-α specifically. The quality of the immunohistochemical method used in the present study is believed to be sufficient for evaluating the distribution of ER-α, since the immunostaining is exclusively nuclear and, in both kinds of negative control sections, immunoreaction is absent.…”

“…The ER-antibody was monoclonal and raised to the N-terminal (A/B) region of the human ER-α receptor (DAKO A/S, Glostrup, Denmark). Western blot analysis of proteins extracted from human endometrium showed that the antibody reacts with a single band of 67 kDa representing ER-α (Saati et al, 1993). Furthermore, the ER-antibody does not crossreact with ER-β (Pettersson et al, 1997).…”

PNL2, a New Monoclonal Antibody Directed against a Fixative-Resistant Melanocyte Antigen

“…The non-immunoglobulinproducing myeloma cell line P3 ϫ 63 Ag8-653 was used as a fusion partner. Fusions were performed using standard techniques (33,34). Supernatants were tested for antibody-binding activity by ELISA and by indirect immunoperoxidase method on frozen sections of human pancreas and tonsil.…”

Oestrogen Receptor Analysis of Paraffin Sections and Cytosol Samples of Primary Breast Cancer in Relation to Outcome After Adjuvant Tamoxifen Treatment

“…Our aim was to investigate these correlations using a newly developed monoclonal antibody against ER, ER-ID5 (13,14), in a breast cancer series, where all patients had been treated with adjuvant tamoxifen. The findings were also related to other prognostic factors, particularly to the histopathological pattern.…”