Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease.

Abstract

Mice of the strain MRL/Mp-lpr/lpr develop a lupus erythematosus-like syndrome that includes the production of autoantibodies specific for nucleic acid-containing cellular components. We have fused spleen cells from such a mouse with the myeloma SP 2/0 and examined the antibodies produced by the resultant cloned hybrid cell lines by using immunoprecipitation and immunofluorescence techniques. Three types of monoclonal antibodies, specific for Sm, DNA, or rRNA, all antigens to which patients who have lupus make antibodies, have been identified. Patient anti-Sm antibody had previously been reported to precipitate five small nuclear ribonucleoproteins that contain U-1, U-2, U-4, U-5, and U-6 RNAs. The monoclonal anti-Sm antibody gives the same immunoprecipitation pattern, providing direct evidence that the Sm antigen resides on all these RNA-protein complexes. Monoclonal anti-Sm antibody will be valuable in deciphering the biological function of these ubiquitous small nuclear RNPs. A simple competition radioimmunoassay using the monoclonal anti-Sm antibody to titer patient sera is also presented. Uses of monoclonal antibodies for the study of autoimmune disease are discussed.The sera of patients and mice that have autoimmune disorders contain antibodies whose presence often results in tissue injury. The mechanisms leading to the development of autoantibodies are not well understood. However, the appearance of specific antibodies in patient sera has been reported to be associated with particular clinical signs of disease (1). Studies with monoclonal antibodies having the same specificity as such autoantibodies could result in a clearer understanding ofboth the mechanisms leading to the formation of autoantibodies and the role of such antibodies in producing disease. Mouse monoclonal antibodies to DNA and ribosomal RNA have been described (2, 3).Autoantibodies also provide potent tools to probe the structure and function ofthe cellular constituents against which they are directed. We have previously used antibodies from the sera of patients who have systemic lupus erythematosus (SLE) to examine the antigens designated Sm, RNP, La, and Ro (4-6). Each of these antigens resides on discrete particles composed of RNA and protein and called either small nuclear ribonucleoproteins (snRNPs) or small cytoplasmic ribonucleoproteins.Anti-Sm has been found to precipitate five different snRNPs, containing the small RNAs U-1, U-2, U-4, U-5, and U-6, from mammalian cell extracts. One snRNP that bears the RNP as well as the Sm determinant appears to be involved in the nuclear splicing of mRNA precursors (7), while cellular roles for the Laand remaining Sm-bearing snRNPs and for the Ro-containing small cytoplasmic RNPs are unknown. The availability of monoclonal antibodies directed against snRNPs would eliminate many uncertainties in experimental interpretation raised by the use of patient sera.Antibodies to Sm are found in certain SLE patients (1), dogs with a lupus-like syndrome (8), and the MRL/Mp-lpr/lpr (MRL/1) and MRL/M...

Human autoantibody secreted by immortalized lymphocyte cell line against the 68k polypeptide of the u1 small nuclear ribonucleoprotein

“…Microtiter plates coated with the 68K polypeptide were incubated first with various dilutions of 2.73 supernatant, then with Su-2E4 antibody. The effect of MAb 2.73 on the binding of Su-2E4 to 68K was monitored by the use of horseradish peroxidase-conjugated anti-human IgM ( p chain-specific) and subseSm MAb Y 12 precipitated the U1, U2, U4, U5, and U6 RNAs ( Figure 5, lane S), and the 2.73 MAb precipitated only the U l RNA ( Figure 5, lane 3), as previously reported (31,36). Since Su-2E4 is of IgM class, modifications of conventional techniques for IgG immunoprecipitation were used (see Materials and Methods).…”

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

“…The Smith antigen precipitates the small nuclear RNA, providing direct evidence that the Smith antigen resides on RNA-protein complexes [20]. The nuclear speckles are rich in pre-mRNA splicing factors and are localized in the interchromatin regions in the nucleoplasm.…”

“…Nuclear speckles were tagged with the Y12 monoclonal antibody (mAb) to Smith antigen (Sm), which recognizes common core proteins of snRNPs involved in RNA processing [20,21]. The Smith antigen precipitates the small nuclear RNA, providing direct evidence that the Smith antigen resides on RNA-protein complexes [20].…”

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

“…The snRNPs were detected by indirect immunofluorescence using the monoclonal anti-Sm antibody Y12 (Lerner et al 1981), kindly donated by Joan Steitz (Yale University, CT, USA). The cells were incubated with a 1 : 500 dilution of this antibody in PBS/Tween 20 (0.05%) at 37°C for 45 min.…”

“…Visualization of major components of the splicing machinery-the U1, U2, U4/U6 and U5 snRNP (small nuclear ribonucleoprotein particles) proteins-by means of indirect immunofluorescence-revealed a wide nuclear distribution and, in addition, a speckled nuclear staining with 20 to 50 speckles consisting of higher snRNP concentrations (Mattioli & Reichlin 1971, Northway & Tan 1972, Lerner et al 1981, Spector et al 1983, Spector 1984, Reuter et al 1984, Bachmann et al 1986, Nyman et al 1986, Verheijen et al 1986. For most of these analyses anti-Sm antibodies were used, which recognize proteins which are common to all splicing snRNPs.…”