Purification and biochemical characterization of nuclear ribonucleoprotein antigen using purified antibody from serum of a patient with mixed connective tissue disease.
We have achieved a high degree of purification of nuclear ribonucleoprotein antigen from calf thymus nuclear extract through antibody affinity chromatography. Antibody to nuclear ribonucleoprotein was purified from the serum of a patient with mixed connective tissue disease and Sepharose 4B was covalently coupled with the purified human antibody. The sodium thiocyanate eluate from the affinity column contained active ribonucleoprotein antigen and the specific activity of the antigen in this eluate was 488 times higher than the original nuclear extract. The protein component of the eluate consisted of six polypeptides determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of them were shown to be antigenic by a hemagglutination inhibition test. The molecular weights of these two peptides were approximately 13,000 and those of the other four were 13,000, 13,000, 30,000, and 65,000. The ribonucleic acid component of the eluate was shown by urea-polyacrylamide gel electrophoresis to contain five polynucleotides. Two of the five, estimated to contain 40 and 60 nucleosides, had antigenic activity. The other three polynucleotides which had more than 77 nucleosides had no antigenic activity. No modified nucleosides were found in these ribonucleic acid molecules. Even in this highly purified ribonucleoprotein antigen, Sm antigen was detected by immunodiffusion. This evidence indicated that there are some molecular associations between ribonucleoprotein and Sm antigens as has previously been suggested.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000302:isolation & purification; D000941:Antigens / Q000302:isolation & purification*; D002467:Cell Nucleus / Q000276:immunology; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D005779:Immunodiffusion; D008947:Mixed Connective Tissue Disease / Q000276:immunology*; D009698:Nucleoproteins / Q000276:immunology*; D009705:Nucleosides / Q000032:analysis; D012261:Ribonucleoproteins / Q000032:analysis / Q000276:immunology*; D013950:Thymus Gland / Q000276:immunology
D000596:Amino Acids; D000906:Antibodies; D000941:Antigens; D009698:Nucleoproteins; D009705:Nucleosides; D012261:Ribonucleoproteins