Development of a radioimmunoassay for human milk-fat-globule membrane glycoprotein. Its quantitation in spent media from both primary and established mammary and non-mammary epithelial cell lines.
A radioimmunoassay (RIA) was developed for the quantitative measurement of a glycoprotein which was purified from human milk-fat-globule membrane (MFGM) and termed MFGM-pg 70. The assay with a sensitivity of detecting 30 ng of MFGM-pg 70/ml was employed to quantitate the levels of MFGM-gp 70 shed in supernatants from primary cultures of normal and malignant human breast cells and from various established cell lines of human mammary including myoepithelial and fibroblast and non-mammary malignant epithelial cells. The RIA for MFGM-gp 70 showed that the amount of antigen shed was much higher in supernatants from normal mammary epithelial cells compared with their malignant counterparts grown in primary culture and with those from established cell lines of malignant mammary epithelial cells. No detectable antigen was found in supernatants from cultures of normal myoepithelial-like cell lines or primary cultures of fibroblast cells from breast, or cell lines of squamous carcinomas of head and neck and tongue, renal cell carcinoma and teratoma. Trypsinization of mammary epithelial cells from both primary and established lines resulted in the release of most of the antigen from the surface of the cells, suggesting the presence of this molecule on the cell's surface. Following trypsinization, approximately 99% of the cells was viable, indicating that the release of the antigen in supernatants was due to shedding and not cell death. The levels of MFGM-pg 70 in spent media were unaffected by the lactogenic hormones such as prolactin or insulin. The RIA for MFGM-gp 70 provides a sensitive and quantitative means to in vitro study the synthesis of a membrane glycoprotein from human mammary epithelium.
D001940:Breast / Q000032:analysis*; D001943:Breast Neoplasms / Q000032:analysis; D002460:Cell Line; D003470:Culture Media; D004848:Epithelium / Q000032:analysis; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007768:Lactalbumin / Q000096:biosynthesis; D008565:Membrane Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D018396:Mucin-1; D011388:Prolactin / Q000494:pharmacology; D011863:Radioimmunoassay
D003470:Culture Media; D007106:Immune Sera; D008565:Membrane Proteins; D018396:Mucin-1; D011388:Prolactin; D007768:Lactalbumin