Preliminary Evaluates The Accuracy Of Using Protein Microarray To Detect Anti-ENA Antibody

[Background]One of the characters of CTD (connective tissue disease) is that there are many kinds of autoantibodies targeting at autoantigens. These autoantibodies are relative with diffusion of immunity mechanism, and clinical performance of the diseases. Furthermore, studies have proved that, in some autoimmunity diseases, there are these autoantibodies in patients' serum several years before the diseases' coming. At present, there are dozens of screening tests or diagnostic tests of the autoantibodies in clinical practice, however there is no uniform method for detecting various autoantibodies, even has two or more methods to detect the same one autoantibody. The variety of these methods' sensitivity and specificity is result in many problems. It often confuses clinical doctors, brings up difficulties to compare the results of different mechanical hospitals, and makes harms to body and economic lost with patients' repeating various tests. Therefore, developing new method to detect autoantibodies is not only a meaningful task to the study of immunity diseases, but also a demanding tool of clinical practice.In clinical practice, there are many traditional methods, such as IBT (Immuno blot test), ID (Immuno diffusion), CIE (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immunosorbent assay), and etc. These traditional methods need too much patients' serum to perform dripping test in different reacting pool. Whereas, protein microarray method differentiates itself from traditional ways, it can fulfill various aims with minim specimen at the same time in clinical practice. So it can reduce cost, and promote efficiency of autoimmunity diseases' diagnoses. This article aims to evaluate the sensitivity, specificity, and detecting accuracy of this method by comparing with traditional methods, ID and ELISA. This article uses this method to detect anti-extractable nuclear antigen antibody of standard serum, specimens of continuous general test serum, specimens of donators' serum, and specimens of diagnosed patients' serum.[Objectives] 1. Preliminary evaluates the accuracy of using protein microarray method to detect anti-ENA antibody;2. Compares the results of ID method and protein microarrary method, and calculates the sensitivity, specificity, and agreement rate of this new way;3. Evaluates the conformity of this method with other traditional methods, such as ID and ELISA;4. Compares the positive rates of these three methods in detecting anti-ENA antibody.[Materials and Methods]Specimens:1. 10 pieces standard serum from Clinical Detecting Center of Ministry of Health;2. 1580 pieces serum of general test from Rheumatologic Department of Peking Union Medical College;3. 300 pieces serum of donators;4. 445 pieces serum of patients, including 22 polymyositis and dermatomyositis patients, 48 systemic selerosis patients, 135 systemic lupus erythematosus patients, 95 primary Sjogren's syndrome, 72 other CTD patients, and 73 non-CTD patients;Methods:1. Protein Microarray method: Using ink jet robotic microarrayer to make purified antigen array on chemical modified 96-well-microtier, then coating, washing, blocking, sample incubating, chemical illuminating, signal identifying, and data processing. Use this method with blind procedure to test standard serum from Clinical Detecting Center of Ministry of Health, and comparing it with the standard results.2. ID method: Using counter immuno-electrophoresis method and immunoblot test (IBT) to do a parallel test, if there is a precipitating line or any strip, do a further classical clubs-hole method test with antigen of extracted fluid from human spleen. Using double blind procedure to compare protein microarray method and ID method, testing 1580 pieces serum of general test from Rheumatologic Department of Peking Union Medical College and 300 pieces serum of donators; 3. ELISA method: The kits of ELISA test are produced by ERUOIMMUN, according its test instruction to do the test. Use these three kinds of methods ( Microarray, ID, and ELISA) to detect 445 pieces serum of patients, and compare their results.[Results]1. Using the method of protein microarray, according to blind procedure, to detect 10 pieces standard serum of anti-ENA antibody, the result is exactly the same with the fact.2. Using the method of protein microarray to test 300 pieces serum of donators, the results are all negative.3. According double blind procedure, using the methods of protein microarray and ID to test 1580 pieces serum of general test, and the results are:1) Using ID as golden standard, the sensitivity of protein microarray method to test anti-Sm anti-RNP, anti-SSA, anti-SSB, and anti-rRNP antibodies is greater than 90% in 95 per cent confidence interval, and the sensitivity to test anti-Sc-70 and anti-Jo-1 antibodies is greater than 85% in 95 per cent confidence interval. Except anti-SSA antibody (84.88%-84.94%), anti-SSB antibody (89.91%-89.95%), and anti-rRNP antibody (89.17%-89.41%), the specificity of all the other antibodies is greater than 90% in 95 per cent confidence interval. And except anti-SSA antibody (86.87%-86.91%), the agreement rate of all the other antibodies is greater than 90% in 95 per cent confidence interval (p<0.05).2) Anti-RNP antibody (κ=0.833), anti-SCL-70 antibody (κ=0.786), anti-Jo-1 antibody (k=0.920), the agreement rate of these two methods is very good (κ>0.75); and the agreement rate is relatively good (P<0.05), when testing anti-Sm antibody (κ=0.423), anti-SSA antibody (κ=0.646), anti-SSB antibody (κ=0.443), and anti-rRNP(κ=0.617).3) ID as golden standard, all the areas of under ROC curves are greater than 0.9 (p<0.05), using protein microarray method to test these seven kinds of antibodies.4) The positive rates of four antibodies, anti-Sm, anti-RNP, anti-SSA, anti-SSB, are 5.89%, 8.40%, 29.67%, 14.12% using protein microarray method, and are 1.65%, 6.83%,18.13%, 4.95% using ID method. The former list is better than the later one (p<0.05). But the positive rates are no difference between the two methods to detect the other three antibodies, anti-Sc-70, anti-Jo-1, and anti-rRNP (p>0.05).4. Comparing the results of three methods1) The agreement rate between protein microarray and ID methods is very good, when testing three antibodies, anti-Scl-70, anti-Jo-1, anti-rRNP (p<0.05). The positive rates are no significant differences between protein microarray and ID, when testing three kinds of serum, SSc, PM/DM, and SLE. The positive rates are 54.2%, 27.3%, 16.7% using protein microarray, and are 62.5%, 31.8%, 11.1% using ID, and p=0.125, p=1.000, p=1.000. The results of protein microarray and ELISA methods are perfectly the same.2) Pertaining to anti-Sm antibody, in SLE group, the agreement rate between protein microarray and ID methods is relatively good (κ=0.631); but the agreement rate between protein microarray and ELISA is very good,κ=0.884 and p<0.05; Pertaining to anti-RNP antibody, in three groups (SLE, pSS, CTD), these three kinds of methods have very good agreement rates,κ>0.7 and p<0.05; Pertaining to anti-SSA antibody, the agreement rate between protein microarray and ELISA is very good,κ>0.7 and p<0.05 in each group; and the agreement rate between protein microarray and ID is relatively good,κis between 0.4and 0.75, and p<0.05;Pertaining to anti-SSB antibody, the agreement rate between protein microarray and ELISA is relatively good,κis between 0.682 and 0.820, and p<0.05; the agreement rate between protein microarray and ID is relatively no good,κis between 0.250 and 0.318, p<0.05;3) Comparing the positive rates of protein microarray with ELISA, there is no difference (p>0.05), when testing anti-Sm, anti-SSA, and anti-SSB antibodies; but the positive rates of these two methods are greater than the one of ID, p<0.05; pertaining to anti-RNP antibody, the positive rates in each group show no difference between protein microarray and ELISA; and except greater than ID in SLE group (p<0.05), the positive rates in other groups of protein microarray remains no difference with ID's.[Conclusions]1. Both sensitivity and specificity of protein microarray method to detect anti-ENA antibody are comparatively higher, and the agreement rates with ID and ELISA are better, so the accuracy is good.2. The sensitivity of protein microarray method is higher than using ID method to test anti-Sm antibody, anti-RNP antibody, anti-SSA antibody, anti-SSB antibody, and is comparatively the same with using ELISA method. And its sensitivity is the same with the other two methods, when detecting anti-Scl-70 antibody, anti-Jo-1 antibody, anti-rRNP antibody.3. The method of Protein microarray has many advantages in clinical screening autoantibodies, such as high throughput, parallel, high speed, high sensitivity, and high specificity. But its testing results of different technical platforms are not conformable, needing further stabilizing the technical platform and much more evaluating processes.