Evaluation of a new automated enzyme fluoroimmunoassay using recombinant plasmid dsDNA for the detection of anti-dsDNA antibodies in SLE

ELISA methods to detect anti-double-stranded DNA (anti-dsDNA) antibodies are highly sensitive, but are less specific for the diagnosis of SLE than the immunofluorescence test on Crithidia luciliae (CLIFT) and the Farr assay because they also detect low-avidity antibodies. This study evaluated the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of a new automated fluoroimmunoassay (EliA dsDNA; Pharmacia, Freiburg, Germany). We compared the results with those obtained using a commercial CLIFT and an in-house anti-dsDNA IgG ELISA method, and verified its putative ability to detect only high-avidity anti-dsDNA antibodies. Sera from 100 SLE patients and 120 controls were studied. The control group included 20 healthy donors, 70 patients with other rheumatic diseases (32 systemic sclerosis (SSc); 18 primary Sj?gren syndrome (pSS), 20 rheumatoid arthritis (RA)), and 30 patients with various infectious diseases (ID). Anti-dsDNA avidity was estimated using an ELISA method based upon the law of mass action, and a simplified Scatchard plot analysis for data elaboration; the apparent affinity constant (Kaa) was calculated and expressed as arbitrary units (L/U). Sensitivity, specificity, PPV, and NPV for SLE were 64%, 95.8%, 93.8% and 72.7%, respectively, for the EliA anti-dsDNA assay; 55%, 99.2%, 98.5%, and 68.8%, respectively, for the CLIFT; and 64%, 93.3%, 90.6%, and 72.3%, respectively, for the in-house ELISA. Although EliA anti-dsDNA was positive mainly in SLE patients with high- (Kaa>80 L/U) and intermediate- (Kaa 30-80 L/U) avidity antibodies (45.3% and 49.9%, respectively), it was also positive in five (7.8%) SLE patients with low-avidity anti-dsDNA antibodies, and five controls (three SSc, one pSS, and one ID) (mean Kaa = 16.4 +/- 9.04 L/U). In conclusion, EliA anti-dsDNA assay showed a higher sensitivity than the CLIFT, and a good specificity and PPV for SLE. Its putative ability to detect only high-avidity anti-dsDNA antibodies remains questionable.     

四种方法检测抗双链DNA抗体诊断效能的对比分析

目的评价绿蝇短膜虫间接荧光免疫法（CLIFT）、酶联免疫吸附法（ELISA）、线性免疫分析法（LIA）和化学发光免疫分析法（CLIA）4种方法检测抗双链DNA（dsDNA）抗体对系统性红斑狼疮（SLE）的诊断效能。方法用4种方法学分别检测2012年7月-2013年6月178例研究对象血清,其中SLE患者86例,其他自身免疫性疾病患者62例,健康体检者30例。结果 4种方法检测抗dsDNA抗体对SLE的诊断效能从高到低依次为ELISA、CLIA、CLIFT和LIA,其中以ELISA的灵敏度最高（67.4%）,而CLIA的特异度最高（95.6%）;3种实验方法（ELISA、LIA、CLIA）与比较方法（CLIFT）的检测一致性非常高（Kappa值均〉0.8）;如果特异度设定为95.0%,ELISA和CLIA的的灵敏度差异无统计学意义（58.1%、60.5%,P〉0.05）。结论 CLIFT、ELISA、LIA和CLIA均可用于抗dsDNA抗体的临床检测,检测结果一致性高,诊断效能与方法学本身和所使用的截断值密切相关。     

ELISA法联合CLIFT法检测抗双链DNA-IgG抗体应用于系统性红斑狼疮的诊断价值

目的：采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）, 观察抗双链DNA（double-stranded DNA, dsDNA）IgG抗体在系统性红斑狼疮（systemic lupus erythematosus, SLE）患者和非SLE患者中的分布特点, 并评估ELISA法联合绿蝇短膜虫间接免疫荧光试验（crithidia luciliae immunofluorescence test, CLIFT）检测抗dsDNA-IgG抗体用于SLE诊断的效能。方法：收集上海交通大学医学院附属瑞金医院检测抗dsDNA-IgG抗体的住院患者的临床资料, 共4 726例采用ELISA法, 其中830例同时采用CLIFT检测, 用受试者工作特征曲线（receiver operating characteristic curve, ROC）分析2种检测方法辅助诊断SLE的效能。结果：采用ELISA法检测的 4 726例住院患者中, dsDNA抗体阳性者398例, 其中SLE 183例, 占dsDNA 阳性者46.0%, 非 SLE疾病依次为肝脏疾病（28.4%）、其他结缔组织病（7.5%）、血液系统疾病（5.3%）及呼吸系统疾病（3.8%）;830例采用CLIFT检测ds DNA, 134例结果显示为dsDNA抗体阳性的患者中, 其中SLE者占94.0%, 另有非SLE诊断病例包括自身免疫性肝病3例, 肝硬化、肾炎、银屑病和抗磷脂综合征各1例。830例完成ELISA法与CLIFT双检测的患者中, SLE患者共197例, 非SLE患者633例, ELISA法检测灵敏度为74.6%, 特异度仅为92.9%;而CLIFT法测dsDNA抗体具有较高的特异度, 为98.7%, 灵敏度为64.0%。联合2种方法平行检测dsDNA抗体可明显提高SLE的诊断效能, ROC曲线下面积最高（0.869 0）。结论：单独应用ELISA法时, 应警惕假阳性较高, 需排除其他非SLE疾病的诊断;联合使用ELISA法和CLIFT法检测抗dsDNA抗体可较单独应用CLIFT法明显提高SLE的诊断效能, 二者联合平行检测结果为阴性, 用于排除SLE更有意义。     

Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for the diagnosis of systemic lupus erythematosus--implications for inter-laboratory testing

BackgroundAnti-double stranded DNA (anti-dsDNA) autoantibodies are considered hallmark of systemic lupus erythematosus (SLE).MethodsTo determine concordance between assays for the detection of this marker, we analyzed 100 antinuclear antibody (ANA) positive sera with a homogeneous pattern and titers ≥ 1:160 by indirect immunofluorescence assay (IFA) on HEp-2 cells, 100 consecutive anti-dsDNA IgG ELISA-negative as well as 100 healthy control samples using six commercial ELISAs and the Crithidia luciliae immunofluorescence test (CLIFT).ResultsThe positivity rates for the ELISAs in the ANA positive group ranged from 55.0 to 88.0% with specificities from 84.0 to 98.0%. The CLIFT had a positivity rate of 68.0% and specificity of 84%. In the previously screened anti-dsDNA IgG-negative group, the positivity rates ranged from 1 to 19%. The overall correlations between the ELISAs ranged from 73.0 to 89.5% and varied from 70.0 to 80.0% among specific ELISAs and CLIFT.ConclusionsOur data show variable degree of concordance between anti-dsDNA IgG ELISAs which may significantly impact inter-laboratory testing as well as the diagnosis and management of SLE patients. Although some of the ELISAs show comparable performance to the CLIFT, the degree of concordance between these assays at high antibody levels suggests that CLIFT is still a relevant confirmatory tool.     

Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus

BackgroundsAmong anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE).MethodsAnti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases.ResultsFarr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE.ConclusionsFarr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment.     

不同方法联合检测SLE患者血清中双链DNA抗体的性能评估

目的探讨双链DNA抗体(dsDNA抗体)在常见自身免疫性疾病中的检出情况,评估不同方法联合检测系统性红斑狼疮(SLE)患者血清中dsDNA抗体的性能。方法使用ELISA法(ELISA1、ELISA2)和间接免疫荧光(IIF)法同时检测300例SLE和495例非SLE自身免疫疾病患者,以及300例健康体检者血清dsDNA抗体,分析单独和联合使用不同方法检测dsDNA抗体对SLE的诊断效能。结果 ELISA1、ELISA2和IIF在SLE患者中的检出率分别为42.3%、35.3%和38.0%,在非SLE自身免疫性疾病患者中最高检出率为1.2%,在健康体检人群中最高为0.3%。在SLE人群,3种方法中ELISA1和ELISA2法,ELISA1和IIF法,以及ELISA2和IIF法联合Kappa值分别为0.672、0.398和0.512(P0.05)。而只有ELISA1和ELISA2间的检测率差异有统计学意义(P=0.003)。ELISA1、ELISA2和IIF法检测dsDNA抗体的受试者工作特征曲线下面积(AUC)分别为0.708、0.672和0.687;ELISA1和IIF法的联合检出率最高为54.7%,AUC为0.764;3种方法同时检测,任何1种方法阳性即判断为阳性时,检出率为57.7%,AUC为0.781。结论联合使用ELISA法和IIF法,可以明显提升dsDNA抗体在SLE中的检出率。     

Complement-fixing anti--double-stranded DNA with the Crithidia method: a better indicator of active SLE than anti-DNA with the Farr method.

Twenty-seven patients with SLE of juvenile onset were studies for 2 years. Episodes of active disease and quiescence were defined and were related to levels of anti-dsDNA and C3. Two methods for the detection of anti-dsDNA--the Farr assay and Cr immunofluorescence--were compared. The latter method was also used to differentiate anti-dsDNA according to its Ig class and its CF property. Positive tests for anti-dsDNA and low C3 levels were correlated with activity of the disease. Results with the Farr assay and Cr Ig test were comparable, but both low C3 and Cr CF anti-dsDNA showed a significantly stronger association (p less than 0.001) with active SLE than did DNA binding by the Farr method or Cr Ig anti-dsDNA. Furthermore, in six patients followed during active disease and remission, a negative Cr CF test was the earliest sign of ensuing clinical remission; DNA binding and C3 levels took weeks to months longer to normalize. This predictive value of CF anti-dsDNA may be useful in monitoring therapy for SLE, especially if symptoms due to prior renal damage may be confused with active disease as in lupus nephritis.     

Cell lines that express membrane-associated DNA for anti-DNA antibody detection

BACKGROUND: In this study, we evaluated the analytical reliability and clinical sensitivity of two tests (indirect immunofluorescence and ELISA) for anti-DNA antibodies which use cell membrane DNA (cmDNA) as antigen (Wil2 NS lymphoblastoid B cell line). METHODS: We tested 97 sera of patients with systemic lupus erythematosus (SLE) and 140 control sera from healthy subjects or from patients with other systemic autoimmune diseases or infectious diseases. The results obtained with the two anti-cmDNA kits were compared with those obtained with three other methods: indirect immunofluorescence on Crithidia luciliae, ELISA anti-double-stranded DNA (anti-dsDNA) and ELISA anti-nucleosome. RESULTS: The diagnostic sensitivity was 85% for immunofluorescence cmDNA and 90% for ELISA cmDNA, i.e., much higher than that of Crithidia (51%) and ELISA dsDNA (71%) methods, and the same as that of the ELISA nucleosome test (85%). The specificity of the cmDNA tests proved lower (92% for immunofluorescence and 87% for ELISA) than that found by anti-dsDNA Crithidia (99%), ELISA (99%) and anti-nucleosome tests (100%). CONCLUSIONS: The results of our study indicate that cmDNA tests may be used as screening tests in patients with suspected SLE. The anti-nucleosome test offers the best diagnostic accuracy overall and can play an important role in the serological diagnosis of SLE.     

Comparison of three anti-dsDNA assays: Performance and correlation with systemic lupus erythematosus disease activity

ObjectiveTo investigate the BioPlex 2200 multiplex immunoassay and Farrzyme ELISA assays as alternatives to the established Farr radioimmunoassay for the correlation of anti-dsDNA antibodies in the assessment of disease activity in systemic lupus erythematosus (SLE).Design and methodsStandard protocols were used to verify analytical performance claims. Anti-dsDNA antibody levels in SLE patient specimens (N = 105) were measured and assessed for clinical performance using manufacturer cut-off limits along with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score.ResultsAssay precision, measurable range and normal reference interval met the manufacturers' stated claims. Agreement between Farr and BioPlex assays was moderate (positive agreement = 62%; negative agreement = 85%; kappa = 0.48), as was agreement between Farr and Farrzyme assays (positive agreement = 56%; negative agreement = 91%; kappa = 0.51). Mean SLEDAI-2K scores differed significantly between the anti-dsDNA positive and negative groups for BioPlex (p = 0.0006), but not Farr (p = 0.11) or Farrzyme (p = 0.34). ROC curve analysis showed a similar area under the curve (AUC) for all three assays (0.76, 0.74, and 0.73 for Farr, BioPlex, and Farrzyme, respectively) in the discrimination of clinically active disease. Furthermore, increased anti-dsDNA levels from BioPlex showed significant correlation with active renal disease. However, results suggested a lower cut-off for the Farrzyme assay for assessment of global disease activity.ConclusionsBioPlex and Farrzyme assays had similar overall agreement with the Farr assay, with BioPlex best reflecting disease activity in SLE patients.     

系统性红斑狼疮患者抗双链DNA抗体检测及其与免疫学指标的相关性

目的探讨系统性红斑狼疮患者抗双链DNA（dsDNA）抗体与免疫学指标的相关性。方法收集77例系统性红斑狼疮患者血清,采用短膜虫间接免疫荧光法（CLIFT）及酶联免疫吸附测定（ELISA）检测血清抗dsDNA抗体、总补体、补体C3、补体C4水平。结果 77例系统性红斑狼疮患者50例抗dsDNA抗体阳性,27例抗dsDNA抗体阴性。抗dsDNA抗体阳性组和阴性组患者血清IgG、IgM、IgA水平的差异没有统计学意义（P〉0.05）,抗dsDNA抗体阳性组患者血清总补体、补体C3、补体C4水平明显低于阴性患者（P〈0.05）。抗dsDNA抗体阳性患者血清抗dsDNA抗体水平与总补体、补体C3显著相关（P〈0.05）。结论抗dsDNA抗体可能通过补体旁路途径而非经典途径抑制补体生成。     

Anti-nucleosome, anti-chromatin, anti-dsDNA and anti-histone antibody reactivity in systemic lupus erythematosus.

Anti-nucleosome (anti-chromatin) antibodies play a key role in the pathogenesis of systemic lupus erythematosus (SLE). The objective of the present study was to determine the clinical significance of anti-nucleosome (anti-chromatin) antibodies, anti-dsDNA antibodies and anti-histone antibodies in patients with SLE in relation to patients with positive nuclear antibodies and healthy controls. We measured anti-nucleosome (anti-chromatin) antibodies, anti-dsDNA antibodies and anti-histone antibodies in 70 patients with SLE, 35 antinuclear antibody (ANA)-positive subjects without autoimmune disease and 35 blood donors. All antibodies were determined by enzyme-linked immunosorbent assay (ELISA). We obtained the receiver operating characteristic (ROC) curve and the area under the curve (AUC) for each autoantibody. Likewise, we obtained the sensitivity, specificity and positive and negative likelihood ratios for each autoantibody. The highest AUC was obtained for anti-nucleosome (0.898) and the lowest AUC for a kit for anti-dsDNA (0.725). Stratification of the control group (ANA-positive subjects without autoimmune disease and blood donors) produced significant changes in the AUCs; all AUCs decreased when ANA-positive patients without autoimmune disease were considered as controls and all AUCs increased when blood donors were considered as controls. These effects were less marked in anti-dsDNA antibodies. We observed discrepancies between kits (anti-nucleosome and anti-chromatin and two for anti-dsDNA). The highest sensitivity for SLE was obtained for anti-nucleosome antibodies (86%) and the highest specificity was obtained for anti-dsDNA antibodies (90%). In conclusion, anti-nucleosome and anti-chromatin kits show different degrees of clinical accuracy due to the cut-off selected by the manufacturer. Once the kits with the best performance and the optimal cut-offs have been selected, anti-nucleosome antibodies and anti-dsDNA antibodies provide similar information in established SLE.     

抗核糖体P蛋白抗体检测在SLE临床诊断中的意义

目的探讨抗核糖体P蛋白抗体(ARPA)检测对诊断系统性红斑狼疮(SLE)的临床意义。方法用ELISA检测79例SLE患者血清、48例其他系统性风湿病患者和52例健康献血员血清中ARPA。同时采用ELISA检测79例SLE血清中抗dsDNA抗体和抗Sm抗体。结果79例SLE患者中ARPA阳性9例,对诊断SLE的敏感性11.4%,特异性100.0%,与疾病对照组和正常对照组相比,结果的差异有显著性(P<0.05)。ARPA、抗dsDNA抗体和抗Sm抗体其中一项阳性的血清有59例,阳性率为74.7%。结论ARPA与SLE具有高度相关性,可用于SLE的临床诊断。联合检测ARPA与抗Sm抗体和抗dsDNA抗体,可以提高血清学诊断SLE的检出率。     

Predictive value of antinative DNA for systemic lupus erythematosus: comparison of radioimmunoassay and enzyme immunoassay

Positive predictive values for the diagnosis of systemic lupus erythematosus (SLE) were determined for two antinative DNA assays: a Farr-type radioimmunoassay (RIA) and a solid-phase enzyme immunoassay (EIA). The study population consisted of all patients whose EIA anti-DNA results were positive (greater than 250 IU) during a 150 day period; in addition all patients whose EIA anti-DNA results were 100-250 IU during the first 25 days of this period were also included. Simultaneous Farr RIA were performed on all sera, and the Crithidia luciliae immunofluorescent assay was also carried out on those with positive RIA and/or EIA results. Diagnoses were established by chart review, and disease activity was scored for SLE patients. Positive predictive values for SLE were dependent on the level of test positivity, and "normal" limits of 10% binding for the Farr RIA and 800 IU for the EIA gave comparable results. Combination with Crithidia luciliae immunofluorescence increased the sensitivity of both assays, especially the EIA. Results of both assays correlated significantly with activity scores in SLE patients.     

毛细管电泳法检测血清中抗双链DNA抗体

目的：建立一种新的检测抗双链DNA（dsDNA)抗体的方法。方法：采用大肠杆菌质粒DNA作为抗原，用荧光噻咪橙衍生标记双链DNA（dsDNA)，利用毛细管电泳激光诱导荧光法（CE－LIF）检测系统性红斑狼疮（SLE）患者血清中的抗双链DNA（dsDNA)抗体。结果：40例SLE患者有23例阳性，占57．5％，25例其他免疫病患者仅有1例阳性；45名正常人全部阴性，与放射免疫法（Farr)和斑点免疫法（DIBA）比较的结果为：CE－LIF的敏感性高于DIBA法，特异性高于Farr法。结论：该法简便，可靠，可用于临床常规检测SLE患者中的抗dsDNA抗体。     

系统性红斑狼疮患者自身抗体联合检测的意义

目的探讨抗核抗体（ANA），抗双链DNA（dsDNA）抗体，抗Smith（Sm）抗体和抗核小体抗体（AnuA）四种自身抗体单项及联合检测在系统性红斑狼疮（SLE）的诊断中的价值。方法测定56例SLE患者，108例其他结缔组织病患者及50例健康人群血清中的自身抗体。以间接免疫荧光法测定ANA，抗dsDNA抗体；以免疫印迹法测定抗Sm抗体，酶联免疫吸附试验（ELISA）测定AnuA。结果56例SLE患者血清自身抗体检测中：ANA、抗dsDNA抗体、抗Sm抗体、AnuA阳性率分别为：91．0％、44．6％、35．7％、64．3％；特异性分别为：70．9％，100％，98．7％，98．1％。在抗dsDNA抗体，抗Sm抗体，AnuA三者中，任何一项检测结果阴性时，其他两种自身抗体均出现一定阳性率，并以AnuA在抗dsDNA抗体、抗Sm抗体阴性时阳性检出率最高，达51．6％和61．1％。三者联合检测阳性率为83．9％。结论在检测的四种自身抗体中以ANA敏感性最高，其他三种特异性均可达97％以上，除AnuA敏感性稍高，其他两项检测值均低于50％。三者联合检测可较大程度提高SLE检测阳性率，并且特异性也与单独检测差异无显著性，且三者具有明显的互补作用。     

Clinical evaluation of a new automated anti-dsDNA fluorescent immunoassay.

The measurement of anti-double-stranded DNA (anti-dsDNA) antibodies is a useful tool for the diagnosis and the follow-up of systemic lupus erythematosus (SLE). Anti-dsDNA antibodies are involved in the pathogenesis of lupus nephritis and they are, specially the high-avidity antibodies, the most specific antibodies associated with SLE nephritis and active SLE. The aim of the present study was to assess the clinical utility of an enzyme-linked immunosorbent assay (EUSA) that utilizes a circular double-stranded plasmid DNA as a nucleic acid source, adapted to an automated fluorescence immunoassay (EliA dsDNA, Pharmacia, Freiburg, Germany). Also, we compared this method with other immunoassays used in clinical laboratories. We have measured anti-dsDNA antibodies in the serum of 179 patients with a positive result for antinuclear antibodies (ANA). Seventy six sera were from SLE patients (14 men and 62 women), and the other 103 sera (from 20 men and 83 women) constituted the control group. This latter group includes nine Sjogren's syndrome patients, six patients with rheumatoid arthritis and 88 with various other diseases, including connective tissue diseases (n=34), hepatopathies (n= 17; 11 primary biliary cirrhosis and 6 autoimmune hepatitis), and 37 patients with nonautoimmune diseases (viral hepatitis, renal disease, diabetes, exanthema and hypertension). Methods used were "EliA dsDNA" (Pharmacia, Germany), "Varelisa dsDNA" (Pharmacia, Germany), Farr (Amersham, UK) and Chritidia luciliae immunofluorescence test (Vitro-Immun, Germany). We assessed sensitivity, specificity, positive predictive value and negative predictive value in the clinical study, and kappa index and scatter plots in the comparative study. The results show a low concordance between methods (kappa < 0.6). The evaluated EliA method shows a very good specificity for SLE (93.2%) and a good sensitivity for active SLE (70.8%).     

抗双链DNA抗体不同检测方法的对比分析及临床应用研究

目的探讨胶体金斑点法(DIGFA)和酶联免疫吸附法(ELISA)检测抗双链DNA(dsDNA)抗体的临床应用价值。方法采用DIGFA法和ELISA法同时检测系统性红斑狼疮(SLE)患者(n=80)及健康对照者(n=50)血清中抗dsDNA抗体。并将ELISA法的定量检测结果与SLE患者的临床指标进行相关分析。结果 ELISA法检测抗dsDNA抗体的特异性、阳性预测值均高于DIGFA法,但敏感性略低于后者。ELISA法检测的抗dsDNA抗体水平与SLE患者疾病活动性指数、血沉、血清C3及24小时尿蛋白定量结果呈明显相关性。结论 ELISA法检测抗dsDNA抗体水平较DIGFA法准确性更高,并且是监测SLE病情转归的重要实验室指标之一。     

A novel epitope on the C-terminus of SmD1 is recognized by the majority of sera from patients with systemic lupus erythematosus.

The SmD1 protein is a specific target for the autoantibody response in SLE. To further analyze this reactivity epitope, mapping was performed with cellulose-bound 13-mer peptides overlapping 10 amino acids (aa). In this initial approach, 4 out of 15 SLE sera recognized more than five overlapping peptides of the SmD1 C-terminus. Therefore, longer oligopeptides of up to 37 aa of this region were generated and probed for as antigens by ELISA. For the SmD1 aa 83-119 polypeptide, there was a striking increase of reactivity with 70.0% positive reactions out of 167 SLE sera. In contrast, 105 healthy control sera were negative, and only 8.3% of sera from patients with other inflammatory diseases (n = 267) exhibited a response, which was of low level only. The anti-SmD183-119 reactivity was significantly higher in anti-dsDNA antibody positive vs. negative sera (P < 0.001) and correlated with disease activity. Four of five human monoclonal anti-dsDNA antibodies also reacted with SmD183-119. The specificity for SmD1 was demonstrated by inhibition experiments and immunization of rabbits with SmD183-119 inducing SmD1-specific antibodies. In conclusion, the SmD183-119 peptide was demonstrated to be an important and highly specific target of the autoimmune response in SLE. The high sensitivity of this ELISA probably depends on a conformational epitope, which appears not to be accessible in the full-size SmD1 protein.     

两个不同厂家自身抗体定量检测试剂盒的优劣评价

目的：评价四川省新成生物科技有限责任公司（A厂家）研制的赛盟自免棆抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）与已上市销售的同类产品的优劣。方法收集2013年6～8月临床确诊为系统性红斑狼疮（SLE）的患者血清50例设为试验组,收集同期体检健康者血清50例设为对照组,分别采用A厂家和德国某知名B厂家生产的抗dsDNA抗体检测试剂盒进行定量检测,对检测结果进行统计分析。结果A厂家的抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）阳性符合率高于B厂家同类试剂,结果差异具有统计学意义（P＜0．05）,两个厂家的阴性符合率差异无统计学意义（P＞0．05）;线性相关性分析r＝0．9848,转换为定性结果后采用Kappa分析,Kappa系数为0．91。结论A厂家生产的抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）和德国某知名B厂家已上市销售的同类产品具有等效性,符合临床应用要求。     

A fluorescence sedimentation assay for dsDNA antibodies

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, ‘Fluoro-Farr’ assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.