Inhibition of adhesion of F9 embryonal carcinoma cells to substratum by a novel monoclonal antibody, TEC-05, reactive with a developmentally regulated carbohydrate epitope

Abstract

Abstract Embryonal carcinoma cells carry on their surfaces carbohydrate antigns that are also expressed in early embryonic cells. We report here the expression and properties of a new developmentally regulated carbohydrate epitope, which is defined by a monoclonal antibody TEC-05. This antibody was generated by immunization of a rat with mouse embryonal carcinoma cells P19S1801A1. By immunofluorescence, the TEC-5 epitope was first detected on 8-cell-stage mouse embryos and was present on all subsequent stages of preimplantation development. Absorption analysis revealed that TEC-5 epitope was expressed only on a limited number of adult mouse tissues. In the direct radioantibody binding assay, TEC-05 reacted strongly with OTF9–63 cells and with some of the mouse embryonal carcinoma cell lines tested. Its reaction with differentiated cell lines was weak or undetectable. In the course of differentiation of OTF9–63 cells induced by retio nic acid, the epitope disappeared with the onset of morphological differentiation. The binding of the antibody to OTF9–63 cells was inhibited to 50% by 10–50 μM N-acetyllactosamine and lactose. Immunolabelling of extracts from OTF9–63 cells separated by sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis revealed that TEC-5 epitope was carried by high-molecular-weight glycoconjugates (molecular weight >100000). Molecules, isolated from [ 3H]-fucose-labelled OTF9–63 cells by indirect immunoprecipitation with TEC-05 antibody, were degraded by extensive pronase digestion or mild alkaline treatment to large carbohydrate chains that were excluded from a Sephadex G-50 column. Direct evidence that TEC-05 antibody bound to embryoglycan was obtained using a modified Farr's assay. The antibody was found to inhibit adhesion of F9 and OTF9–63 cells to substratum. The inhibitory effect, which could be abrogated by lactose, seemed to be specific, because another IgM monoclonal antibody which also binds to embryogly-can had no effect. Combined data indicated that TEC-05 antibody recognizes a carbohydrate epitope which is involved in cell-substratum adhesion of F9 cells and which provides a new marker for structure-function studies of stage-specific embryonic antigens.