Rat transplantation antigens. I. Extraction and partial purification of a soluble antigen

Abstract

A soluble histocompatibility antigen was extracted from normal rat lymphoid cells by exposure to an hypertonic KCl solution. The antigen was assayed by inhibition of monospecific cytotoxic (CT) alloantiserum and shown to have the specificity AgB 4 or Rt H1.1, the major locus private antigen of the dark agouti strain. Yields were ~2000 CT inhibitory units (ID₅₀) per 10⁹ cells. Gel filtration of the extract over Sephadex G-200 resulted in an approximate four-fold purification with a final concentration of 530 ID₅₀ units per mg protein. The major portion of the antigen appeared at the front of the inner bed volume but a minor included peak at about 65,000 mol wt. was also seen. Yields and specificity were greatly increased by (a) not washing the cell suspension before extraction (b) removing excess salt from the extract by dialysis rather than gel filtration (c) removing insoluble nucleic acids from the dialysed extract by ultracentrifugation.