Purification of hnRNP from HeLa cells with a monoclonal antibody and its application in ELISA: detection of autoantibodies.
HnRNP antigen from HeLa cells was purified using a monoclonal antibody (383 IgM) that recognizes heterogeneous nuclear ribonucleoprotein (hnRNP). From extracts of HeLa cells radiolabelled with 32P, this antibody immunoprecipitates relatively large RNAs of heterogeneous size which are synthesized in the presence of actinomycin D at doses which suppress synthesis of ribosomal RNAs (characteristic features of heterogeneous nuclear RNA). In immunoblots, 383 IgM binds to seven polypeptides: one of approximately 23,000 daltons, three between 30,000 and 43,000 daltons which correspond to the known hnRNP polypeptides called A1, A2 and C1, one of approximately 50,000 daltons, and a doublet of approximately 120,000 daltons. These proteins comigrate through sucrose density gradients suggesting that they are physically associated. Thus, 383 IgM appears to define an epitope that is shared among a number of the protein components of hnRNP. This antibody has been used to design a simple and fast protocol which allows the determination of autoantibodies from human sera by ELISA.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D001323:Autoantibodies / Q000032:analysis*; D003609:Dactinomycin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D012332:RNA, Heterogeneous Nuclear / Q000276:immunology*; D012261:Ribonucleoproteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D001323:Autoantibodies; C439788:C1 HNRNP; D000939:Epitopes; C503635:HNRNPC protein, human; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C503633:Hnrnpc protein, mouse; D007075:Immunoglobulin M; D012332:RNA, Heterogeneous Nuclear; D012261:Ribonucleoproteins; C439768:hnRNP A2; D003609:Dactinomycin