Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti‐(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1‐U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti‐(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP‐specific B″ antigen (mol. wt. 28 500). Antibodies to the U2 RNP‐specific A' protein (mol. wt. 31 000) were found in only one serum. The B″ antigen differs slightly in mol. wt. from the U1‐U6 RNA‐associated B/B' antigens and can be separated from this doublet by two‐dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti‐B″ antibody specificity also reacts with U1 RNPs which is due to cross‐reactivity of the antibody with the U1 RNA‐specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti‐A antibodies contained in anti‐(U1)RNP sera, it also shares epitopes with the U2 RNP‐specific B″ antigen.

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A U2 small nuclear RNA-associated protein, designated B", was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled us to isolate cDNA clone XHB"-1 from a phage Agtll expression library. This done appeared to code for the B"protein as established by in vitro translation of hybrid-selected mRNA. Recently, successful attempts have been described to partially characterize cDNA clones of autoimmune antigens or their associated proteins by making use of patients' sera (22, 23). Here we describe the isolation and characterization of a cDNA clone containing the complete coding sequence for the U2 RNP-specific B" protein. MATERIALS AND METHODSBacterial Strains and Phage Agtll Expression Library. Escherichia coli strains Y1089 and Y1090 were purchased from Promega Biotec (Madison, WI). A phage Xgtll expression library (24) was constructed from human KG1 cells as described (25).Antibody Screening. Antibody screening was performed by using the human anti-(Ul,U2)RNP serum V26 (5) essentially as described by Huynh et al. (26).For immunoblotting, bacterial lysates were prepared by quick-freezing isopropyl f-D-thiogalactoside-induced lysogenic bacteria carrying the recombinant phage (26). Protein lysates were fractionated on a NaDodSO4/10% acrylamide gel and then electroblotted onto nitrocellulose. Immunoblots were probed with antisera as described (27). Partial Peptide Mapping. Slices containing A or B" antigens were excised from preparative NaDodSO4/15% acrylamide gels, and proteins were recovered by electroelution (28). Cross-contamination of both samples was checked by immunoblotting with serum V26 and monoclonal anti-A and anti-B" antibodies (29,30). These samples and the recombinant antigen, affinity-purified by using anti-p-galactosidase columns (Promega Biotec), were transferred into sample wells of a NaDodSO4/18% acrylamide gel together with various amounts of Staphylococcus aureus V8 protease. Digestion was allowed to proceed by shutting off the power for 30 min after the samples had concentrated in the stacking gel (31). After electrophoresis, the gel was blotted onto nitrocellulose and epitope-bearing peptides were detected with serum V26. RESULTSIsolation of Clone AHB"-1. A cDNA library in the phage Xgtll expression vector was screened by using the human Abbreviation: snRNP, small nuclear ribonucleoprotein particle.tTo whom reprint requests should be addressed. 2421The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Autoantibodies from a patient with systemic lupus erythematosus, which recognize U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), were used to map B-cell autoepitopes on the U1 snRNP-specific A protein. This protein contains two regions that are highly similar to regions in the U2 snRNP-specific B" protein. A site termed epitope 2 maps in one such region and was found to react with antibodies crossreactive between A and B". A second site, epitope 1, is situated in a proline-rich region that shows no homology with B". This epitope can bind three different autoantibodies with distinct

One-hundred-and-one patients referred because of Raynaud's phenomenon (RP) were prospectively followed for a mean period of 42 months. At presentation they were screened for signs and symptoms of connective tissue disease (CTD) according to a detailed protocol. At presentation 37 patients had primary RP (PRP), nine had RP in combination with vascular occlusive disease (RP-VOD), 25 had one symptom of a CTD (questionable PRP), 13 had two or more symptoms (undifferentiated CTD, UCTD) and 17 had definite CTD. Progression from one of these groups to another was seen in 24 patients and from PRP, RP-VOD or questionable PRP towards a (U)CTD was seen in 19 patients. Patients with one sign of CTD showed a high tendency (56%) to develop CTD. The presence of ANA as detected by immunofluorescence and by immunoblotting at the start of the study was associated with the future development of symptoms of CTD; positive predictive value 65% and 71% and negative predictive value 93% and 83%, respectively. ANA-testing by immunoblotting was of special help in predicting the development of scleroderma, the CREST syndrome and mixed connective tissue disease. In conclusion, testing for ANA by indirect immunofluorescence helps to discriminate between patients with persisting PRP and those who will develop a CTD, while testing for ANA by the immunoblotting technique helps to predict the development of a specific CTD.