The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods

Abstract

In a previous study it was shown that La/SSB contains four linear epitopes, p147-154, p291-302, p301-318 and p349-364. The aim of the present study was to investigate the value of the synthetic epitope analogues of the La/SSB autoantigen for the detection of antibodies to La/SSB, in comparison with recombinant La and fragments of this protein. A total of 122 sera with anti-La/SSB activity, from patients with primary Sjögren's syndrome (pSS) or systemic lupus erythematosus (SLE), were tested in various peptide-based assays. In addition, 62 sera from pSS or SLE patients with other autoantibody specificities and 95 sera from healthy individuals were used as controls. The autoantibody specificity was identified by counter immunoelectrophoresis and immunoblot. The peptide-based ELISA assays presented sensitivities ranging from 78% to 88-8% and specificities from 69% to 94-3%. Dot blot assays exhibited sensitivities ranging from 93-6% to 97%, but remarkably lower specificities from 56% to 88%. The most sensitive and specific peptide 349GSGKGKVQFQGKKTKF364 was synthesized and attached on a tetramer sequential oligopeptide carrier SOC4 and used for immunoassay development. Assays based on the recombinant native La protein, the La-C terminal (215 aa), and the N-terminal of La with a mutation at base pair 640 (nine adenines instead of eight) were also developed and compared with the SOC4 peptide-based assay. Of anti-La-positive sera, 88.1% were reactive with both the synthetic peptide SOC4-(349-364aa) and the recombinant La protein. Eighty-three percent of sera were reactive with the La N-terminus and 67.8% of sera were reactive with the La C-terminus. Using sera that were anti-Ro-positive but anti-La-negative, 37% were reactive with the recombinant protein, 26% with the La N-terminus, 33% with the La C-terminus and only 11 % with the synthetic peptide. Our results suggest that the synthetic peptide epitopes exhibit high sensitivity and specificity for the detection of anti-La/ SSB antibodies in ELISA and dot blot techniques. The peptide SOC4-(349-364aa) has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein.

Sjögren’s Syndrome: Autoantibodies to Cellular Antigens

“…The prevalence of antibodies against several La/SSB epitopes in Greek autoimmune sera is shown in table 2. In the same study it was also demonstrated that the most sensitive and specific peptide (amino acids 349-364) attached on a tetramer sequential oligopeptide SOC 4 has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein [49].…”

Autoantibodies to Extractable Nuclear Antigens: Making Detection and Interpretation More Meaningful

“…Gel-based assays also depend on the precipitation of antigen-antibody complexes. The majority of anti-SS-B antibodies recognize linear rather than conformational epitopes (38). These occur at a low frequency in the SS-B ribonucleoprotein and have low antigenicity, resulting in a reduced ability to precipitate immune complexes.…”

“…The use of eucaryotic expression systems may help resolve this problem (24). Another potential source of pure antigen is synthetic peptides; however, these present solely linear epitopes and may therefore be useful only for antigens such as SS-B, where the major known epitopes are linear (38). The low specificity of ELISAs cannot be overcome simply by increasing the cutoff limits of detection.…”

Key Sequences Involved in the Spreading of the Systemic Autoimmune Response to Spliceosomal Proteins

“…In La, major epitopes have been identified in the RNP motif [56±59]. In particular, B-cell epitope mapping of the human La protein using the peptide mimotope scanning technique showed that anti-La autoantibodies from patients with SS and SLE recognize four major linear antigenic epitopes, spanning the amino acids sequences 145±164, 289±308, 301±318 and 349±364 [59,60]. A certain degree of similarity was observed between the segment 151± 158 of La and the RNP1 motif (Fig.…”