Isolation and characterization of tumour-specific antigen from the serum of rats bearing transplanted aminoazo dye-induced hepatomas.

Tumour-specific antigen has been purified from the serum of animals bearing large progressively growing transplants of an aminoazo dye-induced hepatoma (D23). Antigenic activity was detected by the capacity of serum fractions to neutralize reactivity of antibody in hepatoma immune serum for cell surface expressed antigen on viable hepatoma cells as assessed by using the indirect membrane immunofluorescence test. Serum from animals bearing large i.p. grafts of tumour was fractionated by gel filtration on Sephadex G-150 columns at pH 7.3 and pH 2.8 to yield a crude antigen fraction which was further purified by affinity chromatography by using syngeneic rat hepatoma D23 immune IgG insolubilized on Sepharose 4B. The purified antigen exhibited a pI of approximately 4.6 on analytical gel isoelectric focusing. Molecular weight determination on Sephadex G-200 at pH 7.3 in nondenaturing buffer indicated a primary molecular weight of 55,000 and a small amount of aggregated material (mol. wt. greater than 250,000).