Isolation of membrane‐associated tumour‐specific antigen from an aminoazo‐dye‐induced rat hepatoma

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Abstract
Tumour‐specific antigen on the surface of cells of a transplanted rat hepatoma (D23), originally induced by 4‐dimethylaminoazobenzene, was assayed by its capacity to absorb antibody directed specifically against this component and demonstrable by membrane immunofluorescence tests with viable hepatoma cells in suspension. Quantitative assay of antigen in subcellular fractions of tumour, in comparison with that on intact cells, was obtained by this method, the specificity of antibody absorption being confirmed by the lack of effect of intact cells or subcellular fractions of other rat hepatomas and unrelated tumours.Homogenization of hepatoma cells by nitrogen cavitation resulted in the release of membrane fractions retaining hepatoma‐D23‐specific antigen, the recovery being 13 to 17% of that expressed on intact tumour cells. Antigen was equally distributed between large‐particle (sedimented at 10,000 × g) and small‐particle (sedimented at 105,000 × g) fractions, indicating that the method of cell rupture produced antigen‐associated membrane fragments of varying size. No antigenic activity was detectable in soluble cytoplasmic fractions of the tumour. These studies demonstrate that tumour‐specific antigen can be isolated in membrane fragments with the same individual specificity as that demonstrated by immunofluorescence staining of intact hepatoma cells with immune serum or by the induction of tumour immunity.