Biosensor analysis of beta2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies

BACKGROUND: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-beta2-glycoprotein I (beta2GPI) autoantibodies (anti-beta2GPI) and pathogen-specific beta2GPI cross-reactive antibodies that occur transiently during infections. METHODS: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-beta2GPI in serum samples, affinity-purified human beta2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to beta2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with beta2GPI-specific ELISA. RESULTS: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50-500, resonance units (RU) for anti-beta2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-beta2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized beta2GPI. In contrast to APS patient samples, no significant anti-beta2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection. CONCLUSIONS: The SPR biosensor system enables specific detection of APS-associated beta2GPI-reactive APL and differentiation from beta2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-beta2GPI responses in APS patient sera gives additional information regarding the influence of anti-beta2GPI IgG and IgM isotype distribution.     

Operationally defined single- and double-stranded DNA antigens in the Farr assay: diagnostic value

Abstract. The specificity of the Farr assay for the detection of antibodies to double-stranded (ds) DNA is highly dependent on the properties of the radiolabelled antigen. Since dsDNA is notoriously difficult to prepare free of single-stranded (ss) regions, we have examined the feasibility of using in vitro 125 I-labelled calf thymus DNA separated in predominantly ss and ds fractions upon hydroxyapatite chromatography as antigens in the ammonium sulphate assay. Characterization of these defined antigens showed for the 'ss fraction': a sedimentation coefficient of 7S, molecular weights between 0.05 and 0.25 times 10 6 daltons and the presence of 34% ss-regions. The corresponding values for the 'ds fraction' were: 9.5S, 0.2-1.5 times 10⁶ daltons and 10% ss regions. Using each of these ¹²⁵I-labelled DNA fractions, the ammonium sulphate test was performed on a variety of sera from patients with rheumatic and immunological diseases. The results show that 80% of the sera from systemic lupus erythematosus (SLE) sera have dsDNA binding over 2 standard deviations of the normal mean and that this binding is always higher than that to the ss-antigen. On the other hand, a minority of non-SLE sera gave a slightly abnormal binding with the ds-antigen but in contrast to SLE sera, these always had a higher binding value with the ss-antigen. Furthermore, when the binding of SLE and of these non-SLE sera was tested against a perfectly double-stranded DNA preparation of Adenovirus serotype 2, SLE sera still gave high values, whereas the binding of non-SLE sera was abolished. Taken altogether, these results suggest that these two operationally defined DNA antigens can, when used simultaneously, provide a specific serological diagnosis. These data also support the idea that the presence of true anti-dsDNA binding in a human sera is an exclusive SLE feature.     

Autoantibody-mediated impairment of DNASE1L3 activity in sporadic systemic lupus erythematosus

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.     

An in vitro assay for detection of glomerular binding IgG autoantibodies in patients with systemic lupus erythematosus.

The deposition of anti-dsDNA antibodies in the glomerulus is believed to play a critical role in the pathogenesis of nephritis in SLE. However, an absolute correlation between serum levels of anti-dsDNA antibodies and renal disease has not been found. Recently a glomerular binding assay (GBA) has been developed to detect IgG binding to isolated rat glomeruli. We have used the GBA to study sera from four groups of SLE patients: (A) + anti-dsDNA antibodies, active nephritis; (B) - anti-dsDNA antibodies, active nephritis; (C) + anti-dsDNA antibodies, no nephritis; and (D) - anti-dsDNA antibodies, no nephritis. The serum anti-dsDNA antibodies in group A and group C patients could not be distinguished on the basis of isotype, charge, or cross-reactivity with histones. Nevertheless, the mean intensity of glomerular immunofluorescence was significantly higher in group A than in the three other patient groups and distinguished between patients with serum anti-dsDNA antibodies who had nephritis and those without clinically apparent nephritis. GBA reactivity was unaffected by DNase treatment of sera, but was partially inhibited by preincubation with dsDNA. These findings are consistent with the hypothesis that some anti-dsDNA antibodies cross-react with glomerular components and that the presence of this cross-reactivity is associated with, and may be responsible for, the development of nephritis. In addition, we have identified a group of SLE patients with renal disease and typical renal histopathology and immune deposits who do not have serum anti-dsDNA antibodies or antibodies that directly bind to glomeruli in the GBA. The mechanism of renal immune deposition in these patients remains to be determined.     

Repertoire cloning of lupus anti-DNA autoantibodies.

To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.     

Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation.

Although sunlight is known to induce skin lesions in patients with systemic lupus erythematosus (SLE) and to exacerbate systemic manifestations, the underlying mechanisms remain obscure. We report experiments that show enhanced binding of IgG autoantibodies to the cell surface membrane of ultraviolet-B (UVB) irradiated (200-1,600 J/m2) cultured SLE keratinocytes in 10 out of 12 such cell strains. The autoantibody probes showing increased binding were directed against the soluble intracellular antigens, Sm, RNP, SSA/Ro, SSB/La, whereas serum with anti-dsDNA activity did not demonstrate such binding. Control keratinocytes from several sources shared low level binding of autoantibodies after ultraviolet light exposure. In addition, 4/6 UVB-sensitive SLE strains showed increased autoantibody binding to the surface of SLE keratinocytes after UVA exposure (50-150 kJ/m2), but of lower magnitude. When UVB-sensitive nonirradiated SLE strains were exposed to autologous serum, 3/8 sera demonstrated a striking increase in IgG binding, which increased further after UVB exposure. Enhanced expression of saline-soluble intracellular antigens on the cell surface membrane of patient, but not control, keratinocytes may, in part, explain the photosensitivity of patients with SLE.     

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%).     

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.     

系统性红斑狼疮患者自身抗体联合检测的临床意义

目的：探讨3种抗体联合检测诊断系统性红斑狼疮（SLE）的临床意义。方法选择2012年1月至2014年1月本院收治的SLE患者60例（SLE组）,以同期于本院体检健康者60例作为对照组。采用酶联免疫吸附法检测抗双链DNA（dsDNA）抗体,采用免疫印迹法检测抗核小体抗体（AnuA）、抗Sm抗体,比较3种抗体单独检测及联合检测的诊断效能。结果 SLE组AnuA、抗Sm抗体、抗dsDNA抗体及3种抗体联合检测阳性率分别为93．3％、33．3％、46．7％、96．7％,均高于对照组检测结果（ P＜0．05）。3种抗体联合检测具有更高的S L E诊断效能。结论 AnuA、抗ds-DNA抗体、抗Sm抗体联合检测有助于避免SLE的漏诊,从而达到早期诊断、治疗的目的。     

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。     

抗SmD1、抗核小体抗体、抗双链DNA抗体在系统性红斑狼疮诊断中的意义

目的　评价血清抗SmD1、抗核小体抗体(AnuA)、抗双链DNA(dsDNA)联合检测在系统性红斑狼疮(SLE)诊断中的价值。方法　40例SLE患者、14例其他结缔组织病患者及20名健康人血清抗dsDNA采用放射免疫法检测,抗SmD1、AnuA用德国IMTEC公司抗核抗体谱线性印迹法检测。结果　SLE患者单项检测抗dsD NA、抗SmD1、AnuA的阳性率分别为42. 5%、80. 0%、72. 5%,三者联合检测后的阳性率为87. 5%,诊断效率为85. 2%。结论　抗SmD1、AnuA、抗dsDNA联合检测提高了诊断SLE的敏感性和诊断效率,可避免因单项检测出现的漏诊情况,对SLE的诊断和治疗有重要意义。     

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。     

抗细胞膜DNA抗体在三种自身抗体阴性系统性红斑狼疮中的诊断价值

目的 研究抗细胞膜DNA（mDNA）抗体在抗Sm抗体、抗双链DNA（dsDNA）抗体和抗核小体抗体（AnuA）等特异性自身抗体阴性的系统性红斑狼疮（SLE）中的诊断意义。方法 随机选择145例SLE患者。以Raji细胞为底物，采用间接免疫荧光法检测SLE患者血清抗mDNA抗体和抗dsDNA抗体，采用免疫印迹法检测抗Sm抗体，酶联免疫吸附法测定AnuA。分析抗mDNA抗体在其他特异性自身抗体阴性SLE患者中的诊断价值。结果 抗mDNA抗体在SLE中的阳性率为69．7％，明显高于抗Sm抗体（19．7％）、抗dsDNA抗体（31．9％）和AnuA（45．8％，均P〈0．01）。在抗dsDNA抗体阴性的SLE患者中，抗mDNA抗体的阳性率达64．3％；在抗Sm抗体阴性的SLE患者中，抗mDNA抗体的阳性率为70．2％；而AnuA阴性的SLE患者中，抗mDNA抗体的阳性率为60．3％。结论 抗mDNA抗体是一种敏感性较高的SLE血清学标志之一，尤其对抗Sm抗体、抗dsDNA抗体或AnuA阴性SLE的诊断具有重要参考意义。     

An immunofluorescence assay for double-stranded DNA antibodies using the Crithidia luciliae kinetoplast as a double-stranded DNA substrate.

Circulating antibodies to dsDNA are found predominately in SLE and are seen most often in those patients with active systemic disease, particularly severe lupus glomerulonephritis. An IIF technique for measuring these antibodies has recently been described. This uncomplicated assay employs the kinetoplast of the nonpathogenic hemoflagellate Crithidia lucilliae as a dsDNA substrate. We herein report our experience with this assay, emphasizing the methodology and interpretation of results. Although slightly less sensitive than a radioimmunoassay, we find that this IF technique is a specific and reliable qualitative method for detecting anti-dsDNA. An estimate of the amount of DNA antibody present can be obtained by serum titration. The test was positive in only two patient groups tested, SLE (48%) and MCTD (20%).     

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。     

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.     

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.     

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。     

Clinical significance of ELISA positive and immunofluorescence negative anti-dsDNA antibody

BACKGROUND: Positivity for anti-dsDNA antibody is a diagnostic criterion of systemic lupus erythematosus (SLE). In the present study, the significance of ELISA positive and Crithidia luciliae immunofluorescence test (CLIFT) negative anti-dsDNA sera was evaluated. METHODS: There were 371 consecutive serum samples submitted to for anti-dsDNA testing that were assayed using anti-dsDNA ELISA and CLIFT. Sera showing discrepant results were collected and then examined using 3 commercial anti-dsDNA and anti-ssDNA ELISA kits and by Farr assay. Medical records were reviewed for those patients who were ELISA positive and CLIFT negative for anti-dsDNA. RESULTS: Fifty-two patients of 100 anti-dsDNA ELISA positive patients were negative by CLIFT. For ELISA positive and CLIFT negative sera, Farr assays showed the highest positive rate (72.7%) for the 4 different anti-dsDNA assays (3 commercial kits and the Farr assay). Nearly 80% of 44 ELISA positive and CLIFT negative patients met >or=3 of the SLE classification criteria (excluding the anti-dsDNA criterion). CONCLUSION: Some anti-dsDNA ELISA kits have diagnostic efficiencies that are similar to that of the Farr assay. Moreover, the study identifies a group of patients that are ELISA positive but CLIFT negative for anti-dsDNA, and indicates that the majority of these patients have clinically relevant SLE.     

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.