Mouse monoclonal antibody to embryonic antigen: development, cross-reactivity with rodent and human tumors, and preliminary polypeptide characterization.
Hybridomas producing IgM and IgG monoclonal antibodies (MoAb) to embryonic or fetal antigens (EA) were obtained in a completely syngeneic system. Lethally irradiated, 13-day-gestation, C57BL/6N mouse fetal cells or KCI extracts of these fetal cells obtained from primaparous donors were used as immunogens in several regimens to induce splenocytes in C57BL/6N mice that were utilized to form the hybridomas following fusion with a mouse myeloma line. Successful growth and cloning of the IgM-producing hybridomas required supplementation with factor(s) produced in the growth medium of the macrophage cell line RAW 264.7. An enzyme-linked immunosorbent assay (ELISA) was employed to screen the primary fusion hybridomas for antibody directed against fetal cell or adult cell determinants with the use of freshly explanted tissues. Glutaraldehyde-fixed fetal cells as well as crude fetal cell membranes were used as EA+ target cells (i.e., cell lines known to activate T-lymphocyte-mediated tumor resistance) in a solid-phase ELISA to perform quantitative ELISA adsorption tests of the MoAb. The anti-EA monoclonal IgM and the IgG detected common, embryo-specific antigen(s) on mouse, hamster, and human fetuses. Term fetal cells and adult normal tissues of the mouse, hamster, and human did not express cross-reactive determinants for the MoAb by absorption analysis and/or by direct binding in ELISA. EA expression as oncofetal antigens could also be detected with the monoclones on several rodent tumor cell lines tested as well as on a variety of human carcinomas but not on a spectrum of normal human tissues with the use of indirect ELISA absorption and affinity gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Fluorescence analysis with the monoclones demonstrated specific reactivity with the surface of EA+ tumor cells in the FACS IV flow cytometer. The responsible antigen was carried on a 44- and a 200-kilodalton polypeptide.
W J
WJ
Payne
J H
JH
Coggin
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005260:Female; D005615:Freezing; D006801:Humans; D006825:Hybridomas; D007114:Immunization; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000276:immunology*; D010455:Peptides / Q000032:analysis*; D011247:Pregnancy
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D010455:Peptides; C031192:oncofetal antigens