Molecular characterization of ribonucleoprotein antigens bound by antinuclear antibodies. A diagnostic evaluation

Abstract

The specificity of antinuclear antibodies in sera from 54 patients with various rheumatic diseases was analyzed by immunoprecipitation of "P-or "S-methionine-labeled HeLa cell extracts. Of 35 sera giving a speckled and/or homogeneous immunofluorescence pattern in rat liver nuclei, 20 contained antibodies to nuclear ribonucleoproteins, which are defined by their ribonucleic acid and protein components. Four sera reacted with a nuclear antigen (Ga) which has not been described so far. Of 19 sera with antinucleolar antibodies, 18 did not react with nuclear or nucleolar ribonucleoproteins. Correlation of these molecularly defined antinuclear antibody specificities with immunofluorescence patterns and rheumatic diseases is discussed.Sera with antinuclear antibodies (ANA) have recently been shown to bind nuclear antigens composed of protein and small nuclear ribonucleic acids (snRNAs) (1,2). The technique of immunoadsorption of "P-labeled cell lysates on solid phase staphylococcal protein A and subsequent polyacrylamide gel electrophoresis (PAGE) of the extracted RNA has considerable potential in defining ANA specificities. Characteristic PAGE patterns of snRNAs identify anti-Sm, anti-ribonucleoproteins (anti-RNP) , and antiLa antibodies (2,3), which previously could be identified only by comparison with reference sera reacting with nuclear extracts (4-6).We have analyzed 54 ANA-positive sera from patients with a variety of rheumatic diseases by immunoprecipitation of 32P-and 35S-methionine-labeled HeLa cell extracts. Since this technique allows the definition, independent from reference sera, of certain ANA specificities on a molecular level, disease associations of ANA will be more definitive than with the methods currently used for the characterization of subsets of rheumatic diseases (7). In addition, hitherto unrecognized RNP antigens can be detected and shown to react with sera which may even contain ANA of multiple specificities. Fifty-four sera were selected according to the presence of ANA, regardless of clinical diagnosis. Nineteen had antinucleolar antibodies, and 35 had speckled and/or homogeneous antinuclear antibodies with an immunofluorescence (IF) titer of 1:160 or more. ANA were demonstrated by indirect IF using unfixed rat liver cryostat sections. Antidouble-stranded DNA (anti-dsDNA) antibodies were detected by Farr assay (Amersham). MATERIALS AND METHODS Supported in part byThe antigen used for immunoprecipitation was a 32P HeLa cell extract pre ared by labeling overnight 2 X lo6 5 ml of minimal essential medium without phosphate and 10% dialyzed fetal calf serum. For protein analysis, cells were labeled with 200 pCi of "S-methionine (Amersham, Vmmol). Cells were trypsinized, washed in buffered cells (one small 25-cm 4 flask) with 100 pCi "P-phosphate in

Immunodetection of Poly(A) Binding Protein II in the Cell Nucleus

“…For affinity purification of PAB II-specific antibodies, 10 /.tg of purified PAB II was loaded onto a SDS~polyacrylamide gel and transferred to nitrocellulose. The hlot was stained with Ponceau S, and the band of PAB 11 was cut out and used for affinity purification according to (91. Other antibodies used in this study were a monocional antibody (3A7) directed against the 64-kDa subunit oftbe human cleavage stimulation factor [10], a monoclonal antibody against the splicing factor SC-35 [11], p80 coilin-specific buman autoimmune serum (a gift from Dr. A. Lamond), anti-Sm human autoimmune serum (Küng) [12], and a monoclonal antibody (IGElO) against human PAB I [13].…”

Autoantibodies to double-stranded (ds)DNA immunoprecipitate 18S ribosomal RNA by virtue of their interaction with ribosomal protein S1 and suppress in vitro protein synthesis

“…A procedure based on that of Matter et al [4] was used. MOLT4 cells (2 2 10 6 cells) were labelled with 100 Ci of 32 P-phosphate (ICN Pharmaceuticals Inc., Costa Mesa, CA) in RPMI 1640 medium (phosphate-free) for 16 h. Cells were washed with Trisbuffered saline (TBS), lysed in 0 .…”

Autoantibody to th ribonucleoprotein (nucleolar 7–2 rna protein particle) in patients with systemic sclerosis

“…Analysis of RNAs immunoprecipitated by patients' sera was based on the method described by Matter et al (21). Two milligrams of protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ), preswollen in 500 I.1 of IPP (500 mM NaCI, 10 mM Tris, pH 8.0, 0.1% NP40), was incubated with I0 pl of crude serum for 14 hours, and was washed with IPP.…”