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.