Isolation and characterization of lymphocyte membrane antigen, AgF-1.

A genetically defined, serologically identified antigen of the rat lymphocyte membrane (AgF-1) has been isolated. Viable spleen and lymph node cells, prepared by Ficoll-Hypaque density centrifugation from Fisher rats, were radioiodinated using soluble lactoperoxidase. Extracts, obtained with Nonidet P-40 were shown to contain numerous radio-labeled proteins, including cell-surface globulin. AgF-1 was isolated from these extracts by precipitation with a highly specific alloantibody in conjunction with xenospecific anti-globulin antibody and polyethylene glycol (PEG). The use of PEG greatly increased the efficiency of the double antibody technique. The putative antigenic peak was eluted from sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and specific antigenic activity was recovered. Removal of the SDS from there eluates was achieved by equilibration with urea and passage over an anion exchange resin. Renaturation, as evidenced by specific inhibition of complement-mediated cytotoxicity, occurred upon the removal of urea by dialysis. The molecular weight of the purified antigen was estimated to be 35-40,000 daltons by SDS-PAGE and was unaffected by reduction with 2-mercaptoethanol. Amino acid composition was roughly similar to those reported for the major histocompatibility antigens of the rat and other species.