Immunoelectron Microscopic Localization of Cell Surface Antigens on Rat Hepatocytes Detected with Monoclonal Antibodies (HAM2 and HAM4).

Abstract

The localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2 and HAM4, which recognize the rat major histocompatibility complex (MHC) antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2- or HAM4-immunogold complexes (HAM2-gold and HAM4-gold). Some of the directly stained hepatocytes were further mixed with antimouse IgG-gold complex (IgG-gold) to supplement the direct staining. The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-gold particles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. No different antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells. These results suggest that HAM2 and HAM4 are useful monoclonal antibodies for detecting the localization of the MHC class I antigen and the hepato-renal antigen on the hepatocytes, respectively.