Clinical Value Of Quantitative Assay Of Specific Autoantibodies By Multiplex Bead-based Flow Fluorescent Immunoassay

Objective At present,the methods of detection of autoantibodies are mainly qualitative or semi-quantitative.The quantitative results of specific autoantibodies perform better in reflecting the condition changes of autoimmune diseases than qualitative results.Researches are few on the clinical value of quantitative assay of specific autoantibodies by multiplex bead-based flow fluorescent immunoassay(MBFFI)in autoimmune diseases.In this study,we first compared the results of MBFFI with the results of traditional methods such as immunoblotting and indirect immunofluorescence,and then analyze and evaluate the results of quantitatively detection of specific autoantibodies by MBFFI.We further evaluated its diagnostic value of systemic lupus erythematosus(SLE)and their performance on longitudinal monitoring SLE disease activity.Methods Serum specific autoantibodies(anti-dsDNA,anti-Nucl,anti-His,anti-Sm,antiRNP,anti-Rib P,anti-Scl-70,anti-SSA-60,anti-SSB,anti-PM/Scl,anti-M2,anti-Jo-1,antiCB,and anti-SSA-52)were tested by multiplex bead-based flow fluorescent immunoassay and immunoblot assay in 333 patients with autoimmune diseases(including 114 SLE patients and 219 patients with other autoimmune diseases),44 patients with other diseases and 59 healthy individuals.The consistency of the results was analyzed.Antinuclear antibodies were tested by indirect immunofluorescence assay in all samples.Then sensitivity and specificity of the three methods were compared.We selected seven specific autoantibodies whose percentage of serum level in SLE patients higher than the reference range values were high by MBFFI,and evaluated their utility in SLE diagnosis and monitoring of disease activity.Thirty-eight SLE patients were followed up,and their levels of antibodies in remission after treatment were detected.The changes of the levels of antibodies before and after treatment as well as the correlation between the levels of antibodies and SLE Disease Activity Index(SLEDAI)were analyzed.Results The overall concordance rate between the specific autoantibodies results for all samples by MBFFI and immunoblot was 83.26%(Kappa = 0.655).The concordance rates between the results of the two assays in the autoimmune disease patients ranged from 79.88% to 99.40%,and Kappa coefficients ranged from 0.106 to 0.958.The concordance rates between the results by two assays in healthy controls ranged from 96.61% to 100.00%,and those in the other diseases were from 95.45% to 100.00%.The sensitivity of indirect immunofluorescence in the diagnosis of autoimmune diseases was the highest(P<0.01),and the specificity of MBFFI was the highest(P<0.05).The combination of the two assays improved the sensitivity(P<0.01).The serum level of each antibody was significantly higher in SLE than healthy controls and other autoimmune diseases(P<0.001).The sensitivity of anti-dsDNA was the highest(54.39%).When compared with healthy controls,the specificity of diagnosing SLE ranged from 98.33% to 100.00%.The top five greatest areas under the curve(AUC)of receiver operating characteristic(ROC)were from antiNucl,anti-dsDNA,anti-Sm,anti-RNP and anti-His.When anti-Nucl combined with the other four antibodies,the increased sensitivity was found(P<0.001).When compared with other autoimmune diseases,the specificity of anti-Nucl and anti-Sm was the highest(98.63%).The top five greatest ROC-AUC were from anti-dsDNA,anti-C1 q,anti-Nucl,anti-Sm and anti-RNP.When anti-dsDNA combined with the other four antibodies,the increased sensitivity was found(P<0.001).The serum levels of anti-C1 q,anti-dsDNA,anti-Scl-70,anti-Nucl,and anti-Sm were significantly higher in lupus nephritis(LN)patients than non-LN patients(P<0.05).The ROC-AUC of anti-C1 q was the greatest(0.81)in differentiating LN from non-LN patients,and the sensitivity of anti-dsDNA was the highest(68.97%).When anti-C1 q combined with anti-dsDNA,the sensitivity(74.14%)was higher than the sensitivity of anti-C1q(46.55%)(P<0.001).The decreased serum levels of anti-dsDNA,anti-Nucl,anti-His,anti-C1 q and anti-Scl-70 were found after therapies(P<0.001).The changes in serum levels of five antibodies above were positively correlated with changes in SLEDAI(P<0.05).The changes in the levels of C3 and C4 were not related to the changes in SLEDAI(P>0.05).Conclusion The multiplex bead-based flow fluorescent immunoassay could be a method for specific autoantibody confirmation detection.The combination of MBFFI and indirect immunofluorescence technique(autoantibody screening test)is more suitable for clinical needs.Quantitative detection of anti-dsDNA,anti-Nucl,anti-Sm,anti-RNP and anti-C1 q antibodies by MBFFI was of high diagnostic value for SLE.Combined detection of these autoantibodies can increase the sensitivity of diagnosis.Anti-C1 q might be the best diagnostic marker for LN.Combined detection of anti-dsDNA and anti-C1 q increased the sensitivity of LN diagnosis.The serum levels assay of anti-dsDNA,anti-Nucl,anti-His,and anti-C1 q could reflect the response to the treatments in SLE patients.Anti-Scl-70 antibodies could show a good correlation with SLE disease activity in the context of SLE symptoms and other SLE autoantibodies.They could be more valuable than complements for longitudinal monitoring of SLE disease activity.Predictably,quantitative assay of specific autoantibodies by MBFFI could be more clinically valuable than qualitative assay in the diagnosis,differential diagnosis,treatment monitoring and condition assessment of SLE.