Preferential expression of a Mr 155,000 milk-fat-globule membrane glycoprotein on luminal epithelium of lobules in human breast.

An integral membrane glycoprotein with an apparent molecular weight of 155,000 and isoelectric points ranging from 7.2 to 7.6 has been found to be predominantly expressed on the apical plasma membrane of luminal epithelial cell lining the lobules and terminal ducts in breast. The glycoprotein was purified to homogeneity from human milk-fat-globule membrane and was termed MFGM-gp 155. Polyclonal antibodies were raised which specifically reacted to a single component that electrophoretically comigrated with this glycoprotein in immunoblotting experiments. These antibodies appear to recognize epitopes which are expressed on the protein segment of the glycoprotein. Using an indirect immunohistological method, the glycoprotein was localized predominantly on the apical plasma membrane of luminal epithelial cells lining the alveoli of normal breasts. The expression of the antigen was maintained in both morphologically well- and poorly differentiated lobular carcinoma cells. The antigen was weakly detectable on normal epithelial cells lining the terminal ducts and malignant cells of infiltrating ductal and medullary carcinomas. Expression of the glycoprotein is not organ specific as it is detectable in normal epithelial cells of kidney, pancreas, salivary gland, and stomach and in malignant cells of colon and stomach. The antibodies did not react with large ductal, myoepithelial, stromal, endothelial, epidermal squamous epithelial cells, melanocytes, eccrine sweat glands and ducts, sebaceous glands, erythrocytes, and lymphocytes in breast and skin tissues. Thus, antibodies to this glycoprotein appear to be useful phenotype markers to study differentiation of mammary epithelial cells and the pathogenesis of different histological types of mammary carcinomas, including Paget's disease and signet-ring cell carcinoma of mammary gland.

A novel immunoperoxidase procedure of using human monoclonal antibodies for the detection of cellular antigens in tissue sections.

A sensitive immunoperoxidase method was utilized to detect binding of human monoclonal antibodies to cellular antigens in formalin-fixed and paraffin-embedded tissue sections. The method was a 2-layer system utilizing biotin-labeled human monoclonal antibody and an avidin-biotin-horseradish peroxidase complex (ABC). A comparative study of this and a 4-layer peroxidase-antiperoxidase (PAP) staining method was made. In the PAP system, the monoclonal was followed by rabbit anti-human IgG, swine anti-rabbit IgG as the bridge-antibody and rabbit PAP complex. In both cases, the reaction was visualized by the use of aminoethylcarbazole (AEC) as chromogens. The former procedure was almost as sensitive and, most importantly eliminated an inherent problem associated with the latter namely the difficulty in distinguishing binding of the primary human monoclonal antibody from endogenous human Igs in tissue section. In addition, the 2-stage ABC procedure by comparison requires almost half the time.

Generation and immunohistological characterization of human monoclonal antibodies to mammary carcinoma cells.

The purpose of this study was to identify human antibodies generated against autologous breast tumor cells by the host's immune response. Accordingly, lymphocytes from lymph nodes of seven different patients with metastatic breast carcinomas were immortalized by fusing them with a nonsecreting variant of murine myeloma cells. The screening for binding of antibodies to tumor cells was performed by indirect immunoperoxidase staining of paraffin-embedded tissue sections of the autologous tumor. The selected hybrid cells, after being cloned three times, were stable for the secretion of immunoglobulins for over 2 years. A total of 81 human immunoglobulin-producing clones was obtained from an initial 595 wells with hybrid growth. Nine of these clones produced immunoglobulin M, none of which showed detectable binding to tissue antigens in breast. Seventy-two clones produced immunoglobulin G monoclonal antibodies, and 15 of these showed preferential binding to breast carcinoma cells. Three of these immunoglobulin G monoclonal antibodies were subjected to detailed immunohistological evaluations. Using these antibodies at concentrations ranging from 10 to 100 ng/tissue section, the morphologically normal mammary epithelial cells could be discriminated from their malignant counterparts. The antibodies showed diffuse staining of cytoplasmic components in the malignant counterparts. Under these conditions, lymphocytes, erythrocytes, and stromal cells in breast tissues were unstained. The antibodies showed variable reactivity with malignant epithelial cells of colon and stomach, and with normal epithelial cells lining the renal tubules and sebaceous glands in skin. Antigenic heterogeneity of malignant mammary epithelial cells was revealed. The antibodies may have value in the characterization of tumor-associated antigens responsible for inducing autologous immune responses.

Mouse monoclonal antibody to a melanoma-carcinoma-associated antigen synthesized by a human melanoma cell line propagated in serum-free medium.

A monoclonal antibody, F11, was produced against a tumor-associated antigen from the spent medium of the M14 human malignant melanoma cell line which was grown continuously in serum-free medium. Ouchterlony double-diffusion study revealed that the F11 monoclonal antibody is an immunoglobulin G1. The F11 monoclonal antibody reacted positively with seven of eight (88%) melanoma, five of five (100%) carcinoma, zero to five normal, and zero of two lymphoblastoid cell lines by indirect immunofluorescence test. Also, by indirect immunofluorescence test, F11 monoclonal antibody reacted with cryostat sections from four of five (80%) melanomas, six of seven (86%) carcinomas, zero of one benign nevus, and zero of two benign breast diseases. By the indirect avidin:biotin:peroxidase complex immunoperoxidase method, the F11 monoclonal antibody reacted positively with cryostat sections from five of five (100%) melanomas, five of five (100%) breast cancers, two of two (100%) colon cancers, zero of one benign nevus, and zero of one Hodgkin's disease spleen. Thus, the tumor-associated antigen that the F11 monoclonal antibody recognizes appears to be expressed by melanomas and carcinomas, hence the designation melanoma-carcinoma-associated antigen. Microscopic observations disclosed that the melanoma-carcinoma-associated antigen is present in the cytoplasm, on the membrane of melanoma and carcinoma cells, and in the lumen of glandular structures of breast and colon carcinomas. The molecular weight of the melanoma-carcinoma-associated antigen in spent medium from the M14 CEM cell line is 100,000 as determined by sodium dodecyl sulfate:polyacrylamide gel electrophoretic analysis of indirect immunoprecipitates obtained with the F11 monoclonal antibody.