title: A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera.
abstract: Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.
authors.affiliation: Institute of Immunology and Molecular Genetics, Karlsruhe, F.R.G.
authors.forename: H P
authors.initials: HP
authors.lastname: Seelig
authors.forename: H
authors.initials: H
authors.lastname: Ehrfeld
authors.forename: H
authors.initials: H
authors.lastname: Schroeter
authors.forename: C
authors.initials: C
authors.lastname: Heim
authors.forename: M
authors.initials: M
authors.lastname: Renz
mesh_terms: D000918:Antibody Specificity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D012216:Rheumatic Diseases / Q000175:diagnosis / Q000276:immunology; D012261:Ribonucleoproteins / Q000276:immunology*; D017411:Ribonucleoproteins, Small Nuclear; D012680:Sensitivity and Specificity; D013949:Thymus Extracts; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D013949:Thymus Extracts