Affinity Chromatography of RNA-Containing Preparations of Au Antigen

Presence of RNA in some preparations of Australia antigen has been demonstrated by several groups of investigators (1–3). Evidence was also presented that this RNA was enclosed in a lipid-protein coat which suggested that it was a part of Au particle (1). A direct proof as to the linkage of RNA to Au-specific protein was, however, lacking. This paper describes experiments on the isolation of Au antigen by affinity chromatography. We showed that Au specific protein always fractionated with RNA which indicated that both were parts of a single structure. Materials and Methods. Preparations of Australia antigen (Au)1 antigen were obtained from sera of Au-positive blood donors. The sera were centrifuged in 10-ml portions in superspeed 50 MSE ultracentrifuge for 4 hr at 120,000 × G. The bottom layers of four tubes which contained Au antigen were pooled and separated on 2.8- × 100-cm columns loaded with Sephadex G-200 in 0.05 M phosphate buffer, pH 7.2 in 0.9% NaCl (PBS). Fractions which emerged with a void volume were pooled and concentrated by ultrafiltration. Two preparations of Au antigen were obtained by this technique. One contained 4 mg/ml of protein and 2.5% RNA relative to protein; the other, 3.4 mg/ml of protein and 2.0% RNA. Immunoglobulin fraction of anti- Au-antibody-containing serum of human origin was prepared according to Chase (4). Horse hyperimmune serum against human serum protein (Sevac) and normal human serum were used without prior fractionation.