Preliminary Study On The Recognition Epitopes Of Anti-cyclic Citrullinated Peptides Antibody

Objectives:Rheumatoid arthritis is a common chronic autoimmune disease.The main clinical manifestations of rheumatoid arthritis are synovial joint chronic inflammatory response and joint injury.Anti-cyclic citrullinated peptide antibody detected in70%-80% of RA patients and diagnostic specificity up to 98%.ACPA have appeared10 years before clinical manifestations and can predict clinical outcomes.ACPA Provides Basis for Diagnosis and Treatment of RA Patients.The detection of ACPA is based on antigen-antibody specific binding reactions.Therefore,standardization of antigens is crucial for the detection of ACPA.Protein antigens or peptides are a protein modification process after translation,and have a relationship with the pathogenesis of rheumatoid arthritis and the production of anti-CCP antibody.Studies have found that the citrullinated Epstein-Barr Virus nuclear antigen-1 protein sequence has the epitopes that can be recognized by ACPA.In this study,we established a screening method for the recognition of synthetic antigen epitopes by auto-antibodies.This screening method was used to evaluate the reactivity of the citrullinated EBNA-1 candidate peptide with anti-CCP antibody-positive serum and negative serum.Screening for peptides or combinations of peptides that react optimally with ACPA to provide the best antigen for ACPA detection.Methods:1.The establishment of antigenic epitopes screening method.BBS microtiter plate was prepared as a universal enzyme-linked immunoassay plate.Biotinylated bovine serum albumin was coated with polystyrene microtiter plate,and then add streptavidin to form biotinylated bovine serum albumin-streptavidin(Bio-BSA-SA,BBS)microtiter plate.The BBS microtiter plate as a universal microplate,add N-terminal biotinylated antigen peptide,positive serum,HRP-labeled goat anti-human IgG Successively,to form a solid-phase biotinylated bovine serum albumin-streptavidin-biotinylated antigen peptide-anti-CCP antibody-HRP-labeledgoat anti-human IgG complex,add substrate,to measure the optical density value.Optimal dilutions and optimal reaction times for biotinylated peptides and goat anti-human IgG were optimized to determine the optimal reaction conditions for epitope screening.A methodological evaluation was performed on the established method for screening an autoantibody-recognizing epitope,and the reliability of the method was confirmed.2.Screening of ACPA recognition epitopes.A total of 120 clinical serum samples(including 100 patients sera and 20 healthy sera)were selected for peptide screening.Serum samples were detected using EUROIMMUN anti-CCP IgG kit(EnzymeLinked Immunosorbent Assay),using this result as a gold standard for anti-CCP antibody detection.The selected serum anti-CCP antibody results were divided into anti-CCP antibody-positive group and anti-CCP antibody-negative group according to the results of the EUROIMMUN test.Screening tests of candidate peptides according to the antigenic epitope screening method.The ratio of peptide absorbance to blank absorbance is used to determine the binding strength(P/N ratio)of the peptide to the serum sample.Evaluate whether it has potential as an antigen for the detection of ACPA and screen out the best peptide and peptide for optimal parallelism.Results:1.The establishment of antigenic epitope screening method.Using the prepared biotinylated bovine serum albumin-streptavidin universal microtiter plate for antigen epitope screening assay,the optimal antibody dilution ratio was that the concentration of citrulline antigens was 5.0 ?g/mL,and the dilution ratio of HRP-Goat Anti-human IgG antibody was 1:12,000(2.0 ?g/mL).The coating concentrations of biotinylated bovine serum albumin and streptavidin were 4.0 ?g/mL and 10.0 ?g/mL,respectively.The methodological evaluation results showed that the method has good intra and inter-assay precision and the coefficient of variation is less than 6.0%.Negative and positive control sera have good stability and coefficient of variation less than 5.0%.This method can be used for the screening test of autoantibody recognition epitopes.2.Screening of ACPA recognition epitopes.All five peptides were highly reactivewith serum anti-CCP antibodies.The respective diagnostic performances were as follows: E1 diagnostic sensitivity and specificity were 62.3%,71.2%,AUC(0.695);E2 diagnostic sensitivity(60.7%),specificity(91.5%),AUC(0.817);E3 diagnostic sensitivity(42.6%),specificity(67.8%),AUC(0.588);E4 diagnostic sensitivity(82%),specificity(83.1%),AUC(0.872);E5 diagnostic sensitivity(68.9%),specificity(98.3%),AUC(0.843).The sensitivity and specificity of E4 was the best,the area under the receiver operating characteristic curve was the largest.In the combination of peptides,the combined detection sensitivity and specificity of E4 and E5 were the highest,being 94.4% and 81.7%,respectively.Conclusions:1.The epitopes screening test method using biotinylated bovine serum albumin-streptavidin universal assay method has better detection precision,specificity,sensitivity and stability,and can meet the detection requirements.In addition,The test method can be used to screen a variety of different epitopes(peptides).2.All the five peptides could react with ACPA.Among them,E4 had the best sensitivity and specificity,the area under the receiver operating characteristic curve was the largest,and the diagnostic performance was the best.In parallel peptides,the combined sensitivity and specificity of the E4 and E5 peptides in parallel were the highest,and the combined effect was the best.The E4 synthetic citrullinated EBNA-1peptide or combination of E4 and E5 peptides are most likely used as an antigen for the detection of ACPA.