Purification of an Acidic Nuclear Protein Antigen and Demonstration of its Antibodies in Subsets of Patients with Sicca Syndrome

Abstract

Ha, a soluble nuclear protein, was purified from calf thymus nuclear extract by successive application of fractional ammonium sulfate precipitation (60 to 80% saturation), DEAE-Sephadex chromatography (eluted between 0.26 and 0.38 M NaCl in 0.05 M Tris HCl buffer, pH 7.2), and affinity chromatography utilizing an immunoabsorbent column of Sepharose coupled to IgG from a patient with a high titer of antibody to Ha. The Ha antigen could be iodinated by the method of Bolton and Hunter but not by the chloramine-T or lactoperoxidase methods indicating the absence of tyrosine and histidine. The iodinated material was passed through a Sephadex G-100 column to remove minor contaminants. The final product migrated as a single band in polyacrylamide gel electrophoresis. The ¹²⁵I-Ha antigen was utilized to measure specific antibody by the ammonium sulfate method. Elevated serum-binding capacity was observed in 73% of patients with sicca syndrome in the absence of another connective tissue disease and 85% of patients with sicca syndrome associated with systemic lupus erythematosus. It was infrequent in sicca syndrome associated with rheumatoid arthritis (6%) and in systemic lupus erythematosus without sicca syndrome (3%). Anti-Ha antibodies were not found in patients with other connective tissue diseases or normal controls. Antibody to Ha characterizes a subset of patients with sicca syndrome.