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.

Aimns-To generate new monoclonal antibodies directed against melanoma associated antigens using a new melanoma cell line, KAL. Methods-The melanoma cell line was established in culture from a lymph node metastasis of malignant melanoma. Normal Balb/c mice were immunised with KAL cells. Splenocytes were used for fusion experiments using standard techniques. Hybridoma supernatants were tested for antibody binding activity using an indirect inumunoperoxidase method on frozen sections from KAL tumour cells xenografted onto nude mice and human tonsils. KBA.62 was selected because of its reactivity with melanocytic proliferations on both frozen and paraffin wax sections. Results-On immunoblotting, KBA.62 reacted with three bands of 140, 135 and 128 kD and two weak bands of 88 and 73 kD. In normal human tissues basal melanocytes in the epidermis did not react with this antibody and only occasional labelling of endothelial cells was noted. Of the human tumours, KBA.62 reacted strongly and uniformly with the majority of benign (21/ 21) and malignant (75/86) melanocytic proliferations. Staining was localised predominantly to the cell membrane with little or no cytoplasmic reactivity. Negative staining was observed in the majority of human non-melanocytic neoplasms, the exceptions being some carcinomas (11/89), particularly the well differentiated squamous cell type. This, however, was not thought to present a diagnostic problem. Conclusions-KBA.62 appears to be potentially useful in ascertaining the immunomorphological diagnosis of malignant melanoma in routinely processed paraffin wax sections. (J Clin Pathol 1995;48:826-831)

Using spleen cells from athymic nude mice grafted with Ichikawa tumour, we have generated the monoclonal antibody IND.64, which detects a proliferation-associated nuclear antigen. Immunoblotting analysis with IND.64 showed a double band with apparent molecular weights of 395 and 345 kD. In normal human tissues, the antigen detected by IND.64 was expressed only by the nuclei of proliferating cells, such as germinal centre cells of reactive lymph nodes, cortical thymocytes, the basal layer of the skin, and proliferative compartments of the stomach, small intestine, and colon. IND.64 did not react with cells known to be non-proliferative or to show only a low turnover, such as cells of the kidney, liver, smooth muscle, cardiac muscle, and brain. The expression of this antigen during the cell cycle was determined using two approaches: IND.64 immunostaining of synchronized adult bovine aortic endothelial cells and flow cytometric analysis of double-labelled PHA-stimulated peripheral mononuclear blood leucocytes with a DNA marker and IND.64. The antigen recognized by IND.64 was found to appear in the late G1 phase, and persisted in phases S, G2, and M, but was absent in the G0 and early G1 phases. IND.64 was further investigated in different tumour types to evaluate the correlation between the percentage of IND.64-positive cells (IND.64 index) and the histological grade. In non-Hodgkin's lymphomas, an excellent correlation was found between the percentage of IND.64-positive cells and the cytomorphological grade. In nodular sclerosis and mixed cellularity Hodgkin's disease, a high number of Reed-Sternberg cells were positive with IND.64. The non-lymphoid neoplasms investigated showed a variable percentage of positive cells. IND.64 appears to be a promising tissue marker to complement the evaluation of prognosis in human cancer.