The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

Objectives-To develop an enzyme linked immunosorbent assay (ELISA) using as substrate a synthetic 22-aminoacid peptide, corresponding to the ribosomal P0, P1 and P2 common epitope. To study the specificity and sensitivity of the method and evaluate the frequency and clinical associations of anti-P antibodies in two groups of systemic lupus erythematosus (SLE) patients: (a) unselected SLE patients and (b) SLE patients with central nervous system (CNS) involvement. Patients and methods-The C-terminal 22 aminoacid peptide of the ribosomal P proteins (Lys-Lys-Glu-Glu-Lys-Lys-GluGlu-Lys-Ser-Glu-Glu-Glu-Asp-Glu-AspMet-Gly-Phe-Gly-Leu-Phe-Asp) was synthesised according to Merrifield's solid phase procedure. Purification of the peptide was performed by preparative high performance liquid chromatography and confirmed by amino acid analysis. Using this peptide, in a concentration 5 µg/ml, an ELISA was developed. The presence of anti-P antibodies was evaluated by western blot using purified ribosomal proteins from rat liver. Sera from 178 consecutive patients with SLE and 28 patients with SLE and CNS manifestations were tested. Sera from 58 patients with rheumatoid arthritis and 57 patients with primary Sjögren's syndrome were used as controls. The cut oV point of the assay was defined using 124 normal sera. Results-The specificity of the assay was evaluated by homologous inhibition. Pretreatment of positive sera with soluble 22mer peptide of the ribosomal P proteins resulted in 88% inhibition. The concordance between the peptide assay and western blot was found to be 83%. Thirty three of 178 (18.6%) of the unselected SLE patients had antibodies to P-protein common epitope. Their presence was associated with more active disease (European Consensus Lupus Activity Measurement, ECLAM scoring system) (p<0.001), higher levels of anti-ds DNA antibodies (p<0.05) and lower levels of the C4 component of complement (p<0.01). Eleven of 28 (39.3%) patients with SLE and active CNS involvement had antibodies to P-protein.The overall prevalence of anti-P antibodies in active CNS disease patients was statistically significantly higher, as compared with unselected SLE patients ( 2 =6.04, p<0.05). These antibodies were found in a high proportion of patients without anticardiolipin antibodies (52.4%) and they were associated with diVuse CNS involvement (psychiatric disorders (71%) and epilepsy (75%)). Conclusions-A synthetic analogue of the common epitope of ribosomal P-proteins can be use as an antigen for the detection of anti-P antibodies. These antibodies are associated with active SLE and CNS involvement particularly in patients without anticardiolipin antibodies. (Ann Rheum Dis 2000;59:99-104) Autoantibodies to ribosomal P proteins (anti-P antibodies) were recognised several years ago as a distinct group of autoantibodies directed against the P 0, P 1 , and P 2 proteins located on the larger (60 S) subunit of the eukaryotic ribosomes.1 2 Sera containing anti-P antibodies react preferentially with a common epitope consisting ...

SUMMARYThe B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 364 . Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147 HKAFKGSI 154 presented 83·3% similarity with the 139 HKGFKGVD 146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147 HKAFKGSI 154 to 100% to epitope 349 GSGKGKVQGKKTKF 364 . The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93·6% and 85·6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147 HKAFKGSI 154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.

Anti-Ro60KD autoantibodies are commonly found in sera from patients with primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). In order to identify the epitopes of this autoantigen, 22-mer, synthetic peptides overlapping by eight residues, and covering the entire sequence of the Ro60KD autoantigen were prepared. Three groups of sera were evaluated according to their autoantibody specificites. The first group consisted of monospecific anti Ro60KD sera from four patients with SLE and one with SS, the second one was composed of anti-Ro60KD+anti-La(SSB)-positive sera from four patients with SS and the third group included three normal sera and one anti Ro52KD serum. It was found that sera from SLE patients interact with a common antigenic site spanning the sequence TKYKQRNGWSHKDLLRSHLKP (169-190) of the Ro60KD protein. On the other hand, sera from SS patients recognise the ELYKEKALSVETEKLLKYLEAV (211-232) region of this autoantigen. Determination of the minimal required peptide length for optimal antibody recognition showed that the defined epitopes can be shortened to the NGWSHKDLLR (175-184) and KALSVETEKLLKYLEAV (216-232) sequences respectively. Inhibition experiments using the Ro60KD antigen and soluble peptides corresponding to the 175-184 and 216-232 segments further confirmed the specific antibody binding. These results, although only a small number of sera were used, indicate that the Ro60KD autoantigen, which is not characterized by disease specificity, contains two discrete epitopes specifically recognized from SLE and SS patient sera. Finally, the sequence similarity of the NGWSHKDLLR (175-184) epitope with some of the HLA haplotypes, associated with anti-Ro response, deserves to be noted.

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.

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.

Background: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. Materials and Methods: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308aa and 349-364aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(abЈ) 2 fragments from anti-peptide IgG were prepared and F(abЈ) 2 -IgG interactions were evaluated using a specific anti-idiotypic ELISA. Results: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(abЈ) 2 bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and crossinhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative-anti-Sm/RNP positive sera from patients with SLE. Conclusions: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.

Objectives: To investigate the clinical and immunogenetic aspects of antibody formation against Ro/SSA and La/SSB as well as their linear B cell epitopes in patients with primary Sjögren's syndrome (pSS) from different European countries. Patients and methods: Ninety patients with pSS from six European centres were studied. Serum samples from all patients were tested in a control laboratory for anti-Ro/SSA and anti-La/SSB autoantibodies by RNA precipitation assay and autoantibodies to the previously reported B cell linear epitopes of Ro 60 kDa (p169-190aa and p211-232aa) and La/SSB (p147-154aa, p291-302aa, p301-318aa, and p349-364aa). DNA from 88 patients was used for the determination of HLA-DRB1, -DQA1, and -DQB1 genotypes. Analysis of the results was performed in the 88 patients who were genotyped and tested also for antipeptide antibodies. Results: Antibodies to B cell epitopes of Ro 60 kDa were detected at a low frequency (range 10-37%). In contrast, B cell epitopes of La/SSB were detected frequently (range 58-86%) among the anti-La/SSB positive sera. Autoantibodies to the La/SSB epitope, p349-364aa, were significantly positively associated with longer disease duration (p<0.05), recurrent or permanent parotid gland enlargement (p<0.005), and a higher proportion of non-exocrine manifestations (p<0.005), compared with patients without autoantibodies. The presence of anti-Ro/SSA and anti-La/SSB autoantibodies was significantly associated with the presence of HLA-DRB1*03 and DQB1*02 (p=0.038 and p=0.034, respectively). This association was even more prominent and extended to HLA-DQA1*0501 when patients were stratified according the presence of autoantibodies to discrete La/SSB B cell epitopes in comparison with autoantibody negative patients (p<0.01). They were found also to be highly associated with the alleles HLA-DQB1*02 and HLA-DQA1*0501 as well as the presence of a shared amino acid motif in the region 59-69aa of DQB1 first domain (p<0.01, respectively). Conclusions: Autoantibodies against La/SSB, binding to four synthetic peptides, derived from the sequence of the La protein were identified with increased frequency in sera of patients with pSS. The formation of autoantibodies against B cell epitope analogues of La/SSB in European patients with pSS may be dependent on the presence of a permissive HLA-DQ heterodimer, most prominently represented by the HLA-DQA1*0501/DQB1*0201 heterodimer, suggesting that a model of HLA restricted presentation of La/SSB peptide determinants is crucial for the autoimmune response against La/SSB.