Preparation of Aminoazo Dye Induced Rat Hepatoma Membrane Fractions Retaining Tumour Specific Antigen

Abstract

Summary.-Membrane fractions were isolated from homogenates of an aminoazo dye induced rat hepatoma (hepatoma D23) by sucrose density gradient centrifugation in zonal rotors. The membrane fractions retained tumour specific antigenic determinants and exhibited an increased antigenic activity over other subcellular membrane fractions, as defined by their capacity to quantitatively neutralize the membrane immunofluorescence staining of viable hepatoma D23 cells by antibody in tumour immune serum. In contrast, no antigenic activity was found to be associated with purified hepatoma D23 nuclei or nuclear membranes as evaluated by the in vitro antigen assay.The two methods described for the isolation of hepatoma D23 membranes have been developed for the large scale fractionation of tumour homogenates in order that further studies upon the nature and immunogenicity of membrane associated tumour specific antigens may be resolved using defined membrane preparations of increased antigenic activity.

Resistance of the Meth A sarcoma-associated rejection antigen to inactivation with glutaraldehyde

“…Rogers et al (1980). Prepared according to Price & Baldwin (1974 The present findings are in accord with the current view that the Meth A sarcomaassociated rejection antigen is a soluble protein which is expressed intracellularly (Dubois et al, 1980). The antigen must show some expression at the cell surface, to initiate immune responses and to function as a target for immunological recognition and attack, though how many determinants are required to participate in such reactions is unknown.…”

Immunogenic Properties of Rat Hepatoma Subcellular Fractions

“…These findings are comparable with the data already published on the immunogenicity of crude hepatoma membrane fractions (Baldwin, Embleton and Moore, 1973b) where the principal response was the development of humoral antibody, with a weak cell mediated reaction, and rats did not reject a subsequent challenge with viable tumour cells. In the present studies, subcellular fractions (nuclei and soluble cytoplasmic protein) which are considered to be lacking in hepatoma D23 specific antigen as assessed by in vitro assay (Baldwin and Moore, 1969;Price and Baldwin, 1974) did not elicit antibody formation in treated rats, indicating that these two methods for antigen detection showed good correlation.…”

Epitope analysis of monoclonal antibody NCRC-11 defined antigen isolated from human ovarian and breast carcinomas

“…Tumour tissue from specimens of ovarian mucinous adenocarcinoma, ovarian serous cyst adenocarcinoma and breast carcinoma was homogenized in phosphate buffered saline, pH 7.3 (PBS) containing 5 mM MgCl2, at 4 mlg 1 tissue, and an extra-nuclear membrane (ENM) preparation was isolated as the 105,000g pellets of 600g supernatants of the homogenate (Price & Baldwin, 1974). NCRC-l 1 antibody reactivity with these ENM preparations was confirmed firstly using an ENMantibody binding assay (Price et al, 1986) and secondly by demonstrating that NCRC-1 1 antibody bound to high molecular weight antigens (>400,000 -as reported previously -Price et al, 1985) transferred by electroblotting to nitrocellulose paper from sodium dodecyl sulphate polyacrylamide gels (Towbin et al, 1979) and stained with peroxidase-linked rabbit anti-mouse Ig and diaminobenzidine.…”