Membrane antigens in chronic active hepatitis: Physicochemical and immunological studies of soluble liver homogenate fractions

Abstract

Since the liver membrane components termed liver-specific protein (LSP) and liver membrane antigen (LM-Ag) have been implicated as target antigens in chronic active hepatitis (CAH), we performed physicochemical and immunological analyses of these and other fractions obtained from liver homogenates. Supernatants were prepared from homogenates of human and rabbit liver by centrifugation at 105,000 g for 1 hr; after gel filtration on Sepharose 6B, the LSP fraction was obtained as the excluded peak I and LM-Ag as an included fraction, peak IIB. Comparison by immunodiffusion with reference LSP and guinea pig antisera to LSP confirmed the similarity of our fractions to reference preparations. Upon SDS-polyacrylamide gel electrophoresis (SDS-PAGE), human and rabbit peak I were found to contain at least 25 distinct protein bands with a wide range of molecular weights (14,000->100,000) and the other peaks were found to be equally heterogeneous. Thin-layer polyacrylamide gel isoelectric focusing (PAGIF) revealed 35–40 separate bands in both peaks I and IIB with a wide range of isoelectric points (3.5–10.0). These results indicate that liver homogenate fractions such as LSP and LM-Ag can no longer be considered as single protein preparations, and are in fact highly complex mixtures of multiple proteins. Further separation and characterization of such fractions will facilitate identification of liver antigens for use in in vitro immunological assays.