Detection and identification of significant ANAs in previously determined ANA negative samples

BACKGROUND: An antinuclear antibody (ANA) substrate transfected with the cDNA to hyperexpress the 60 kD SS-A/Ro antigen (HEp-2000) has been shown to detect anti-SS-A/Ro antibodies missed by standard HEp-2 and other immunoassays. Despite this evidence, many laboratories remain convinced that with experienced technicians, standard HEp-2 is acceptable for ANA detection. AIM: To challenge the ability of HEp-2000 to detect anti-SS-A/Ro antibodies in samples previously determined to be ANA negative using standard HEp-2. METHODS: Three hundred and seventy-one pre-screened "negative" ANA samples were provided by a university hospital laboratory in Germany. These samples were tested on the HEp-2000 substrate at a dilution of 1:40 by indirect immunofluorescence (IIF). Samples that screened positive for a nuclear pattern were titered (range of 1:40-1:640) and all ANA-positive patterns were identified. Samples containing at least one positive ANA pattern at a dilution greater than or equal to 1:160 were further tested. Samples that produced a speckled pattern were tested for antibodies to the extractable nuclear antigens (ENA) and samples that showed homogeneous staining were tested for antibodies to dsDNA, and if negative, were then tested for anti-histone antibodies. RESULTS: Ninety-one patient samples were positive with titers > or =1:160. Speckled patterns were the most common finding (30 samples) followed by speckled/homogeneous mixed patterns (19 samples) and samples demonstrating the SS-A/Ro pattern (16 samples) either alone or in combination with other ANA patterns. The remaining 26 positive samples consisted of various other ANA patterns. The most commonly identified ENAs were SS-A/Ro (14 samples), Scl-70 (11 samples) and SSB (6 samples). No antibodies to dsDNA were identified in 23 positive samples with homogeneous staining patterns, though 17 of these samples tested positive for antibodies to histone. CONCLUSIONS: HEp-2000 detected anti-SS-A/Ro antibodies in 16 (4%) of the "ANA negative" samples. In addition to improved sensitivity for anti-SS-A antibodies, HEp-2000 demonstrated improved sensitivity over standard HEp-2 substrate for other significant ANAs including anti-Scl-70, anti-histone, and anti-SS-B antibodies.     

3种检测方法在诊断自身免疫性疾病中的价值

目的通过对抗核抗体、可提取性核抗原(ENA)及抗核抗体谱3检测方法的分析比较,探讨有益于临床诊断的检测组合。方法采用人喉癌上皮细胞及猴肝作为基质的间接免疫荧光法检测抗核抗体;应用欧蒙斑点法检测可提取性核抗原中的如下抗原:nRNP/Sm、Sm、SSA、SSB、Scl-70、Jo-1;以绿蝇短膜虫为基质,应用间接免疫荧光法检测抗双链DNA抗体;通过欧蒙印迹法检测15种自身抗体。结果将3种稀释度(1:10、1:80、1:100)抗核抗体检测的阳性率进行比较,1:10及1:80稀释的血清抗核抗体阳性率差异有统计学意义(P0.01),其余2种比较差异无统计学意义;在核点型、胞浆颗粒型及均质型等多种核型的检测中抗核抗体谱3的阳性检出率高于ENA谱;在抗双链DNA抗体的检测中间接免疫荧光法的阳性检出率略高于欧蒙印迹法。结论在应用间接免疫荧光法的抗核抗体检测中,使用1:80的稀释度更有利于提高检出率,而且涵盖15种抗原的抗核抗体谱3也为临床诊断提供了有利的依据。     

An enzyme immunoassay screening test for the detection of total antinuclear antibodies.

The enzyme immunoassay antinuclear antibody (EIA ANA) screening test method is a new assay format for the qualitative determination of antinuclear antibodies (ANAs) in human serum or plasma. This assay method collectively detects ANAs against double stranded DNA (dsDNA), SS-A/Ro, SS-B/La, Scl-70, Sm, and Sm/RNP antigens, along with serum positive for peripheral, homogeneous, speckled, nucleolar, and centromere patterns. This assay correlated well with data obtained with hemagglutination tests for specific ANA antigens, with the indirect immunofluorescence (IFA) ANA HEp-2 test, and with the Crithidia luciliae IFA test for anti-dsDNA. This new test procedure is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples.     

Diversity of autoantibodies in avian scleroderma. An inherited fibrotic disease of White Leghorn chickens.

University of California, Davis line 200 White Leghorn chickens develop an inherited progressive fibrotic disease that includes the appearance of antinuclear antibodies (ANA). To further characterize these ANA, serial aged line 200 birds were studied. Greater than 50% of line 200 birds develop antinuclear and anticytoplasmic antibodies; fluorescent staining patterns included cytoplasmic spider web, most prevalent at 1 mo of age, and fine speckled patterns, characteristic of chickens 6 mo and older. By enzyme-linked immunosorbent assay, 40.4% of line 200 birds were found to have antibodies to single-stranded DNA (ssDNA). In contrast, antibodies to histones, RNA, or poly A . poly U were not detected. Precipitating antibodies to saline extracts from chicken liver were noted in 33.3% of line 200 birds. Saline extracts from turkey, pheasant, and partridge liver but not rat, rabbit, or mouse tissues were also positive in immunodiffusion testing with these line 200 birds. The antigenicity of chicken liver extracts was sensitive to pronase, protease K. and pH variations greater than 10 and less than 5; however, they were resistant to trypsin. DNase. RNase, and incubation at 37 degrees C and 56 degrees C for 1 h. Cell fractionation in conjunction with column chromatographic techniques revealed that several protein antigens with apparent molecular weights in the range of 62,000-290,000 were present in cytoplasm but not in isolated nuclei. Line 200 sera were not reactive against nuclear ribonucleoprotein, Sm, Scl-70, or SS-B/La antigens. Thus, line 200 chickens develop antinuclear and anticytoplasmic antibodies at an early age, which recognize a unique group of protein antigenic determinants found only in avian species. Moreover, and of particular interest, the presence of autoantibodies to saline-extractable antigens correlated with positive ANA, antibodies in ssDNA, and to the clinical expression of disease.     

Evaluation of a line immunoblot assay for detection of antibodies recognizing extractable nuclear antigens

Sera (n = 90) giving positive results in a screening test for antibodies to extractable nuclear antigens (ENAs) were tested in a line immunoblot assay that measures antibody reactivity with individual ENAs in a single test field. Results were then compared to those obtained in monospecific ENA antibody enzyme immunoassays (EIAs). Discordant results were resolved by immunodiffusion. Of 540 result pairs (90 sera tested for 6 ENAs [Sm/RNP, Sm, SSA, SSB, Scl-70, Jo-1]), 509 (94%) showed concordance. Immunodiffusion resolved 28 of 31 discordant result pairs in favor of the immunoblot result. After resolution of discordant data, the immunoblot assay exhibited 100% sensitivity for all ENA antibodies except those recognizing Scl-70, for which the sensitivity was 89%; specificity was over 96% for all 6 ENA antibodies. These findings show that a line immunoblot assay for the characterization of ENA antibodies yields results comparable to those obtained using monospecific ENA antibody EIAs. The immunoblot assay is easier and less expensive to perform due to its utilization of a single test field. J. Clin. Lab. Anal. 12:320–324, 1998. © 1998 Wiley-Liss, Inc.     

系统性硬皮病抗核抗体谱的检测意义

目的探讨抗核抗体（ANA）和抗核抗体谱（ANAs）对系统性硬皮病的诊断意义。方法分别采用间接免疫荧光法（IIF）和免疫印迹法（IB）对64例SSc患者和30例健康对照者血清中6种自身抗体进行测定。结果IIF检测ANA结果显示,SSc患者中ANA的阳性率为98．4％（63／64）,其荧光染色模型主要为均质核仁型;IB检测6种ENA结果显示,SSc患者以抗Scl-70抗体、抗SS-A抗体和抗nRNP抗体为主,阳性率分别为65．1％（41／64）,29．7％（19／64）和6．3％（4／64）。结论多种自身抗体同时检出,可能提示SSc患者同时伴随有其他的自身免疫性疾病,或者有发生其他自身免疫性疾病的危险;此外,IB法对ANAs检测的阳性率和特异性均较高,对SSc的诊断帮助很大。     

Antinuclear, anticentromere and anti-ScL-70 antibodies in rheumatic diseases]

Methods are described that are used for the titration of antinuclear, anticentromere, and anti-Scl-70 antibodies in systemic scleroderma, systemic lupus erythematosus, and rheumatoid arthritis: indirect immunofluorescence with various antigenic substrates (sections of fresh-frozen rat liver and Hep-2 cell culture), counter-current immunoelectrophoresis, isolation of Scl-70 antigen. Use of Hep-2 cells as a substrate for indirect immunofluorescence was found clinically and diagnostically more effective since it permitted the detection of anticentromere antibodies and anti-Scl-70. Nucleolar, mottled, homogeneous, marginal immunofluorescence types were observed when rat liver sections and Hep-2 cells were used for substrates. Anticentromere antibodies and anti-Scl-70 were isolated significantly more frequently in systemic lupus erythematosus or rheumatoid arthritis.     

Diagnostic assays for Anti-PM/Scl IgG antibodies: Heterogeneity in antibody response or lack of standardization?

BackgroundThe aim of this study was to compare the correlation between new diagnostic methodologies for detecting anti-polymyositis/scleroderma (anti-PM/Scl) IgG antibodies associated with myositis and/or systemic scleroderma assays with existing platforms.MethodsSera from 164 samples previously tested for anti-PM/Scl IgG antibody by immunodiffusion, ID; 171 sera screened for anti-PM/Scl IgG by immunoprecipitation, IP; an additional group of 215 sera tested by ID and 46 healthy blood donor sera were retrospectively evaluated. Anti-PM/Scl IgG antibodies were measured using three PM/Scl-100 specific enzyme immunoassays (EIAs), PM1-alpha (PM1-α) EIA and a line immunoblot assay (LIA) for anti-PM/Scl-75 and ? 100 IgG antibodies. Selected samples were tested for the presence of antinuclear antibody (ANA) by indirect fluorescent antibody (IFA) assay.ResultsThe overall agreement between ID and all anti-PM/Scl IgG EIAs as determined by Crohnbach's alpha was unacceptable (α < 0.50). The concordance between the IP and either LIA or PM1-α EIA was greater than 90% however, the best agreement was seen between the IP and LIA PM/Scl-100 assays (98.3%). Compared to the LIA PM/Scl-75 and PM1-α tests, the LIA PM/Scl-100 IgG assay showed the best specificity in the healthy control group.ConclusionsOur results demonstrate considerable differences between assays for detecting anti-PM/Scl IgG antibodies which cannot be attributable to heterogeneity in antibody response alone. Further characterization and standardization of these assays are needed.     

系统性硬皮病抗核抗体谱的检测分析

目的 通过对系统性硬皮病（SSc）患者抗核抗体谱（ANAs）检测结果的分析,探讨这些抗核抗体（ANA）对系统性硬皮病的诊断价值。方法 分别采用间接免疫荧光法（IIF）和免疫印迹法（IB）对62例SSc患者和20例健康对照者血清中15种抗核抗体进行测定。结果 IIF检测ANA结果显示,SSc患者中ANA的阳性率为98.4%（61/62）,其荧光染色模型主要为均质核仁型;IB检测15种ANA结果显示,SSc患者中的ANA以抗Scl-70抗体、抗SS-A抗体和抗Ro-52抗体为主,在46例弥漫性硬皮病（dSSc）患者中抗Scl-70抗体的阳性率高达67%,特异性为100%。结论 多种自身抗体同时检出,可能提示SSc患者同时伴随有其他的自身免疫性疾病,或者有发生其他自身免疫性疾病的危险;此外,欧蒙免疫印迹试剂盒,对ANAs检测的阳性率和特异性均较高,对SSc的诊断帮助很大。     

抗核抗体谱检测对弥漫性结缔组织病的临床诊断价值

目的通过抗核抗体谱检测结果对弥漫性结缔组织病患者临床诊断进行评价。方法回顾分析246例弥漫性结缔组织病和160例非弥漫性结缔组织病住院患者自身抗体检测结果。结果在各种弥漫性结缔组织病中抗核抗体谱阳性检出率间差异有统计学意义,在系统性红斑狼疮患者中,抗dsDNA抗体阳性率为44.1%,抗Sm抗体阳性率为47%;在混合性结缔组织病中抗UIRNP抗体阳性率达96.4%;在干燥综合征患者中抗SSA和抗SSB抗体阳性率为68.2%和59.1%;在系统性硬化病患者中抗Scl-70抗体阳性率为41.4%;重叠综合征抗dsDNA、抗Sm、抗UIRNP、抗SSA抗体的阳性率为45.4%、18.2%、45.4%、18.2%。结论抗核抗体谱检测对各种弥漫性结缔组织病有重要的鉴别诊断意义。      

Detection of autoantibodies to ss-a/ro by indirect immunofluorescence using a transfected and overexpressed human 60 kd ro autoantigen in hep-2 cells

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Seventy-three sera having SS-A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp-2 cell substrate that had been transfected with a full-length cDNA encoding a human 60 kD SS-A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS-A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty-nine of 73 (95%) SS-A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti-SS-A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS-A/Ro antibodies by IIF on standard HEp-2 substrates. We conclude that SS-A/Ro autoantibodies can be detected by an IIF assay using a HEp-2 cell substrate transfected with a SS-A/Ro cDNA. This new substrate detects SS-A/Ro antibodies that were not identified on standard HEp-2 substrates and by other immunoassays.©1995 wiley-Liss, inc.     

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA)

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep-2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement. For SS-B antibodies, there was a good concordance between all four methods used (Kappa 0.66–0.74). For anti RNP antibodies, the results for CIE/ELISA (Kappa 0.60) were consistent as were the two immunoblot methods (Kappa 0.69). For anti Scl-70 (topoisomerase I) antibody, results from the ELISA and CIE methods were totally consistent (Kappa 1.00). In spite of the high prevalence of anti SS-A/Ro antibodies, the agreement between the methods was poor, without statistical significance. Finally, for Sm antibodies, more consistent results were obtained between CIE/ELISA (Kappa 0.51) and between one of the immunoblotting methods and ELISA (Kappa 0.54). In conclusion, CIE concurs mostly with ELISA for anti- RNP, Scl-70, Sm and SS-B antibodies, but with some disagreement for SS-A antibodies. J. Clin. Lab. Anal. 11:388–392, 1997. © 1997 Wiley-Liss, Inc.     

Scl-86, a marker antigen for diffuse scleroderma.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.     

The optimization of techniques complex of serologic diagnostics of connective tissue systemic diseases

The connective tissue systemic diseases originate from pathologic process following with antinuclear antibodies emergence. To detect these antibodies a significant number of diagnostic tests and techniques has been applied. Besides that, there is no conventional algorithm of antinuclear antibodies diagnostic. To detect antinuclear antibodies a two-fold diagnostic algorithm was applied In the capacity of screening techniques the indirect immunofluorescence technique was applied to the cells of line Hep-2 (antinuclear factor) and detection of antibodies to extractable nuclear antigen. The second stage of diagnostic included the detection of content of more specific antinuclear antibodies using the Lineblott method and the double-helical DNA antibodies. The blood serum from 981 patients with suspected connective tissue systemic diseases, 115 patients with systemic lupus erythematous and 57 healthy individuals was analyzed. The levels of antinuclear factor, nuclear antigen antibodies and double-helical DNA antibodies were detected. The antinuclear factor was detected in 84% and 86% of cases, double-helical DNA antibodies in 55% and 39% of cases depending of reagents using in detecting these characteristics. Among healthy individuals, antinuclear factor was detected in 5% (1/20) of blood serum samples in titers less than 1:160. In the group of patients with suspected connective tissue systemic diseases, antinuclear factor was detected in 48% (474/981) of cases and extractable nuclear antigen in 20% (326/981) of cases. The Lineblott test was positive in 33% (326/981) of patients with suspected connective tissue systemic diseases. Among antinuclear factor positive patients nuclear antigen antibodies were detected in 36% (171/474) and the Lineblott test was positive in 63% (298/474) of cases. Among antinuclear factor negative patients but positive under anti-nuclear antigen identification, the Lineblott test was positive in 6% (28/507) of cases. The two-fold algorithm of nuclear antigen testing is an effective technique to be applied in the clinical diagnostic laboratory. The results of effectiveness of this algorithm demonstrated that this method can ensure 33% of cost savings of testing individuals with higher incidence of diseases.     

125例SLE患者血清抗核抗体及抗核抗体谱检测结果分析

目的探讨抗核抗体（ANA）及抗核抗体谱对系统性红斑狼疮（sLE）的临床价值。方法对1525例确诊的SLE患者血清ANA及ANA谱进行检测分析。ANA和抗双链DNA抗体采用间接免疫荧光法（IIF）,ANA谱采用欧蒙印迹法。结果①125例SLE患者血清ANA阳性共121例,阳性率为96．8％。②患者血清ANA谱大多为抗体二联及以上共同出现阳性,各抗体阳性率分别为抗一ul—nRNP／Sm：48．8％、抗一Smtl5．2％、抗一SS—A：36％、抗一R0—52：54．4％、抗一SS—B：14．4％、抗一scl—70：O．8％、抗一dsDNA：55．2％、抗一Neukleosmone：39．2％、抗一Histone：37．6％、抗一Rib—Pro!：12％、抗一Jo一1和抗一CENP—B未检出。结论ANA和ANA谱联合检测在SLE诊断中具有互补性,可提高检出率,对SLE的诊断分型、治疗、预后判断和病情随访等均有重要的临床价值。     

蛋白芯片技术及免疫印迹技术检测抗核抗体的比对研究

目的 通过比对免疫印迹法与蛋白芯片法对血清标本中抗核抗体的检测结果,验证这两种方法是否具有等效性. 方法 分别用免疫印迹技术和蛋白芯片技术检测70例临床标本的抗核抗体,记录检验结果,并对检查结果进行统计学分析. 结果 经配对资料卡方检验分析,两种方法测定SSA、SSB、SM、Scl-70、Jo-1和CENP时结果差异无...     

Anti-PM/Scl-100 and anti-RNA-polymerase III antibodies in scleroderma

BackgroundSystemic sclerosis (SSc) is a rare autoimmune disease characterized by the presence of various autoantibodies, including anti-centromere, anti-topoisomerase (Scl-70), anti-PM/Scl-100, and anti-RNA-polymerase III (RNA Pol-III) antibodies. Recently, new ELISA based immunoassays have become available for the detection of anti-PM/Scl and anti-RNA Pol-lII antibodies.ObjectiveWe studied the prevalence and clinical association of anti-PM/Scl-100 (PM1-Alpha) and anti-RNA Pol-III antibodies.MethodsAntibodies to PM1-Alpha and RNA Pol-III were measured by ELISA (DR. Fooke Laboratories and Inova Diagnostics, respectively) in 242 patients with various connective tissue diseases (CTD) (including 70 SSc patients) and in 36 non-CTD controls.ResultsLow levels of PM1-Alpha antibodies were found in various CTDs, whereas high levels were exclusively found in SSc, dermatomyositis and polymyositis, albeit at low frequency (4.7%). Anti-RNA Pol-III antibodies were found in 7% of SSc and in 1% of non-CTD and CTD controls. Anti-centromere and anti-Scl-70 antibodies were found in 37% and 21% of SSc patients, respectively. Anti-centromere antibodies were associated with limited cutaneous SSc and anti-Scl-70 antibodies with diffuse cutaneous SSc and interstitial lung disease. Because of the low number of samples positive for anti-PM/Scl-100 or RNA Pol-III antibodies, no clinical feature was statistically correlated with the presence of either reactivity, but taken together the presence of either antibody was correlated with interstitial lung disease. Anti-PM1-Alpha and anti-RNA Pol-III antibodies were mutually exclusive with anti-Scl-70 antibodies.ConclusionsAt high levels, anti-PM/Scl-100 antibodies were associated with SSc, PM, and DM, albeit at low frequency. Anti-RNA Pol-III antibodies were associated with SSc (in 7%) with high specificity.     

类风湿性关节炎患者自身抗体的检测及应用

目的探讨抗核抗体（ANA）检测在类风湿性关节炎（RA）诊断中的意义。方法随机选取该院2010～2013年收治的RA患者67例,以间接免疫荧光法及免疫印迹发检测ANA,以速率散射免疫比浊法检测类风湿因子（RF）,并分析ANA和RF在诊断RA中的相关性。结果检测ANA有利于RA的诊断。67例标本中抗-nRNP／Sm、抗-SS-A、抗一组蛋白阳性率最高,分别为21％、18％、18％。RA组ANA核型主要为均质型,其次为颗粒型,也有部分为阴性。结论临床上治疗RA患者时,应注意监测是否同时患有多重自身免疫性疾病。     

Autoantibody formation in burn patients after inhibition of suppressor T cell activity with polymyxin B

After thermal injury, treatment with polymyxin B blocks suppressor T cell activity by uncoupling endotoxin-mediated T cell activation, but the effect on autoantibody formation is unknown. We examined the presence of antinuclear antibodies to native DNA; to soluble antigens Ro/SSA, La/SSB, Sm, nRNP; and to antiepithelial antibodies in 12 burn patients before and after treatment with polymyxin B and in 24 samples from control burn patients. Low titer antinuclear antibody activity was detected in 25% of pretreatment and 78% of posttreatment samples (p less than 0.01) and in 16.7% of control patients. One polymyxin B-treated patient had a significant antinuclear antibody titer both before and after treatment. Antiepithelial antibodies were detected in 16.7% of early polymyxin B-treated samples and 11.1% of late samples (p less than 0.05) but were also present in 20.8% of controls. Antibodies to native DNA, Ro/SSA, La/SSB, Sm, and nRNP were not detected in any sera.     

Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: comparison with immunofluorescence assay in 307 patients

BACKGROUND: A new enzyme immunoassay (EIA) for automated detection of antinuclear antibodies (ANAs) uses a mixture of HEp-2 cell extracts and multiple recombinant nuclear antigens immobilized on beads. We compared this EIA and an immunofluorescence (IF) assay in a large group of patients and controls. METHODS: We studied 492 healthy individuals and 307 patients with connective tissue diseases (CTDs). Sera were tested by an automated EIA (COBAS Core HEp2 ANA EIA; Roche Diagnostics) and IF. Samples were also tested for eight disease-specific antibodies, including antibodies against U1RNP, Sm, SSA/Ro, SSB/La, Scl-70, Jo-1, dsDNA, and centromere. RESULTS: Areas under ROC curves for the EIA were greater than (P = 0.008-0.012) or numerically identical to areas for the IF method for each of six CTDs studied. ROC areas for EIA were 0.98 (95% confidence interval, 0.95-0.99), 0.99 (0.96-1.00), and 0.99 (0.98-1.00) in systemic lupus erythematosus (n = 111), systemic sclerosis (n = 39), and mixed connective tissue disease (n = 33), respectively. For all 258 CTD patients with conditions other than rheumatoid arthritis (RA), the sensitivity and specificity of the IF method at a cutoff dilution of 1:40 were 92% and 65%, respectively, vs 93% and 79% for the EIA at a cutoff of 0.6. For the IF method at a cutoff dilution of 1:160, sensitivity and specificity were 81% and 87%, respectively, vs 84% and 94%, respectively, for the EIA at a cutoff of 0.9. For 207 sera containing at least one of eight disease-specific ANAs, positivities for the EIA and the IF method were 97.1% and 97.6%, respectively, at cutoffs of 0.6 and 1:40 (P = 0.76). CONCLUSIONS: An EIA that can be performed by a fully automated instrument distinguishes CTDs (except RA) from healthy individuals with both higher sensitivity and specificity than the IF method when the cutoff index was set at 0.9. Moreover, it can be used to exclude the presence of disease-specific ANAs by setting the cutoff index at 0.6 with almost the same efficacy as the IF method.