Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomas: formal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2.
Trop-2 is a cell surface structure recognized by the 162-46.2 mAb and expressed by most human carcinomas. Since the 162-46.2 mAb works poorly in immunoprecipitation, to characterize the structure of Trop-2 we searched for other mAbs directed against this molecule. Selection of candidates was performed by analyzing the characteristics of mAbs directed against epithelial cells and by comparing the staining pattern of each mAb with the one of the 162-46.2 on frozen sections of human epidermis. Two mAbs, T16 and MOv-16, were selected for further analysis. Formal proof that candidate mAbs reacted with Trop-2 was obtained by comparing their binding patterns to mouse L cells transfected with the Trop-2 gene by genomic DNA transfection and selected by FACS using the FITC-162-46.2 mAb. In immunofluorescence FACS analysis the FITC-T16 and FITC-MOv-16 mAbs specifically stained Trop-2 transfectants. The specificity of binding was confirmed by selective blocking of the staining by the respective unconjugated mAb. Interestingly, cross-blocking studies indicated that the 162-46.2, T16 and MOv-16 mAbs recognize the same epitope or closely spaced ones on the Trop-2 molecule. T16 and MOv-16 efficiently immunoprecipitate Trop-2 from Trop-2 transfectants and from the human cell line OVCA-432, indicating that it is a cell surface glycoprotein, with an apparent molecular weight of 57 kD in non-reducing conditions. A weaker band of 38 kD is often co-precipitated with the 57 kD form in an apparently specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Alberti
S
S
Istituto di Ricerche Farmacologiche Mario Negri-Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
Miotti
S
S
Stella
M
M
Klein
C E
CE
Fornaro
M
M
Menard
S
S
Colnaghi
M I
MI
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification; D002277:Carcinoma / Q000276:immunology*; D011232:Chemical Precipitation; D004817:Epidermis / Q000276:immunology; D006801:Humans; D014162:Transfection
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface