Determination Of Autoimmune Response Based On Mimic Epitope Of Cyclic Citrullinated Peptide And It's Meaning In Rheumatoid Arthritis

Objective:In order to study humoral immune reaction and cellular immune reaction of rheumatoid arthritis(RA) patients with cyclic citrullinated peptide(CCP).We used enzyme linked immunosorbent assay (ELISA) for anti-CCP antibody,enzyme-linked Immunospot assay (ELISPOT) and MHC peptide tetramer flow cytometry to detect CCP autoreactive T lymphocyte(CCP-ART) in peripheral blood monouclear cells(PBMC) of RA patients.Then evaluating the correlation between anti-CCP antibody and CCP-ART with clinical symptom and commonly used serology index.And analyzing the value of experimental results for RA diagnoses or evaluates patients' pathogenetic condition.Methods:By using pattern of streptavidin pre-immobilization and conjugation with biotin-CCP(SA/bio-CCP),we successfully optimized the experimental conditions of ELISA for anti-CCP antibody,meanwhile evaluated its methodology and results of application in clinical diseases. Using ELIspot and MHC peptide tetramer flow cytometry to detect CCP autoreactive T lymphocyte which peripheral blood mononuclear cells(PBMC) secrete IFN-γor IL-4 and CD4⁺ CD8^- CCP-Tetramer⁺ cells. Evaluating the value of them for RA diagnosis and clinical application and then analyzing their correlation of them with patients' clinical situation or other correlated serology index.Results:Anti-CCP antibody diagnostic sensitivity,specificity, positive/negative predictive value,positive/negative likelihood ratio and Youden's index in rheumatoid arthritis(RA) patients was 69.4%,96.8%, 89.1%/94.1%,19.28/0.317 and 0.658 respectively.Parallel investigation showed that absorbance and cut-off ratio of anti-CCP antibody was significantly associated(n=61,r=0.92531,p＜0.001 ) with that of Euroimmun assay.Among 168 cases,there had 60 positive and 105 negative simultaneously which tested by both methods,the percent of its result consistency is up to 98.2%.So,it is highly consistent with that of Euroimmun assay(Kappa=0.961,P＜0.001).Area under receiver operative characteristic curve,i.e.AUC^ROC is 0.901,this result suggest that diagnostic accuracy of anti-CCP antibody tested by established method was up to a higher level.Of among 13 early RA cases,there have 10 cases positive(positive rate is 76.9%).There have no evidence that anti-CCP antibody is associated with that of clinical symptom,activity index(ESR,CRP) and RF levels in RA patients.RA patients which their PBMC secrete IFN-γspots forming cells (SFC) is 236.8±40.4 while secrete IL-4 spots forming cells is 11.8±1.8 by ELIspot.The ratio of PBMC secrete IFN-γand IL-4 SFC is 25.8±5.6,which significant higher than other autoimmune disease groups and health controls(P＜0.05).RA patients with anti-CCP antibody positive which their PBMC secrete IFN-γSFC is 288.3±54.8 while patients with anti-CCP antibody negative is 151.5±31.3 and SFC in health controls is 20.8±8.7.There are significant difference between them(P＜0.05).The SFC of IFN-γor IL-4 and absorbance value of anti-CCP antibody has a significant correlation,correlation coefficient(r) is 0.318 and 0.322 respectively(P＜0.05).There are not conclusive correlations between specific SFC number(include IFN-γand IL-4) with RA patients' ex-joint symptom,ANA,CRP and ESR.The correlation between specific SFC numbers and RF is significant,coefficient correlation is 0.563(P＜0.05).AUC^ROC is 0.864 and 0.911 respectively while use SFC numbers of IFN-γand their ratio(IFN-γ/IL-4) in RA diagnosis.There are 20 of 32 RA patients which with high CCP-ART numbers while using 15.1 as cut-off value.The number of RA patients which with anti-CCP antibody positive is 19,while one of them positive is 26.We optimize the MHC peptide tetramer flow cytometry for detect CD4⁺CD8^-CCP-Tetramer⁺cells and consider it as CCP-ART.The number of CCP-ART of RA patients is 1.03±0.76,which significant higher than other groups(P＜0.05).RA patients with anti-CCP antibody positive which their number of CCP-ART is 1.24±0.82,which is significant higher than RA patients with anti-CCP antibody negative, while the latter is significant higher than health controls(P＜0.05).There is a good concordance between the number of CCP-ART and the result of ELIspot.The correlation of CCP-ART with the value of anti-CCP antibody and RF is significant(P＜0.01 or P＜0.05).There are not conclusive correlations between specific SFC number with ANA,CRP and ESR.The number of bossed and pained joint or the pecent of ex-joint symptom of RA patients has the elevatory tendency with the number of CCP-ART cells but no statistic difference.In spite of this,there are 31.4%patients with RF negative have a high number of CCP-ART.We found that the number of CD4⁺ CD8^- CCP-Tetramer⁺ cells is with according to patients' clinical symptoms and RF value change while we observe two RA patients for a long time.AUC^ROC is 0.824 while use MHC peptide tetramer flow cytometry to detect CCP autoreactive T lymphocyte for diagnosing RA.The high number of CCP-ART cells has a potential diagnose and application value for RA diagnose.Conclusions:This work has successfully applied in anti-CCP antibody determination based on amplified system of biotin-streptavidin, and provided the sensitive,specific as well as reliable assay results.So,it is suitable for routine laboratory diagnosis of RA.It will be much cheaper than import reagent.Using CCP to stimulate the PBMC of RA patients and then detect CCP-ART with ELIspot and MHC peptide tetramer flow cytometry.The CCP specific Th1 /Th2 cytokine disequilibrium exists in RA patients and Th1 is predominance.The number of CCP-ART cells in RA is much higher than other autoimmune disease patients and health controls.There has a potential diagnosis and application value for RA especially for RA patients with RF negative.Detecting CCP-ART cells combine with ELISA of anti-CCP antibody can increase sensitive for RA diagnoses and it will profit reasonable evaluate the state of RA.