Isolation and characterization of nuclear ribonucleoprotein complexes using human anti-nuclear ribonucleoprotein antibodies.

We have investigated the feasibility of using human autoimmune antibodies to isolate and characterize specific nuclear ribonucleoprotein (nRNP) complexes. High titers of anti-nRNP antibodies occur in a syndrome called mixed connective tissue disease. IgG antibodies from two mixed connective tissue disease patients were used to construct affinity columns to isolate the antigenic complexes from rat liver nuclei. A maximum of 2.9% of the nuclear RNA and 0.7% of the protein bound to anti-nRNP but not to control IgG columns. A fraction of the bound antigen, comprising less than 0.15% of the total nuclear protein, was isolated in antigenitally active form. The protein moiety of this fraction consisted of two quantitatively major polypeptides of molecular weights 30,000 (P30) and 13,000 (P13). Antigens isolated from both anti-nRNP columns possessed essentially these same two polypeptides. lmmunological tests of crude and purified antigens against anti-nRNP sera from a total of four patients provided additional evidence that antibodies from different individuals are directed against the same nRNP antigen. There were no polypeptides in the isolated antigen which corresponded in molecular weight to the core proteins of heterogeneous nuclear ribonucleoproBin (hnRNP) particles described by other investigators. The binding of both RNA and protein to anti-nRNP columns was greatly reduced by treating the crude antigen with pancreatic RNase A before chromatography. In particular, the binding of P13 was reduced to a fraction of the pre-RNase control. None of the four sera in this study contained antibodies to DNA, histones, RNA, DNA oh&tone complexes, or nonhistone chromosomal proteins.