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Objective To explore the relationship between gut microbiota and anti-dsDNA antibody in systemic lupus erythematosus(lupus)TC mice. Methods ELISA was performed to detect serum anti-dsDNA antibody in the lupus TC mice. Then,the feces of both dsDNA-positive and -negative mice were collected, and 16S rRNA of the stool sample was sequenced by Illumina HiSeq 2500 high-throughput sequencing,to analyze the relationship between gut microbiota and anti-dsDNA antibody level in the two groups. Results The result of ELISA and mouse fecal high-throughput sequencing showed that the species diversity of gut microbiota in the dsDNA-positive TC mice was significantly lower than that in the dsDNA-negative TC mice. The gut microbiota of TC mice in the two groups showed significant differences at different classification levels: Chloroflexi at phylum level, Betaproteobacteria, Deltaproteobacteria and Ktedonobacteria at class level,Burkholderiales,Desulfovibrionales and Ktedonobacterales at order level,Alcaligenaceae and Desulfovibrionaceae at family level,and Parasutterella and Desulfovibrio at genus level(P<0.05 for all). Conclusions The flora composition of gut microbiota in lupus TC mice is highly correlated with anti-dsDNA antibody. The results of our study provide a basis for further elucidation of the relationship between gut microbiota and the pathogenetic mechanism of systemic lupus erythematosus.
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Objective To explore the verification methods for the performance of quantitative detection kit of anti-dsDNA antibodiy with enzyme-linked immunosorbent assay (ELISA).Methods The precision was verified according to the EP15-A2 document approved by the American Clinical and Laboratory Standards Institute(CLSI).The accuracy was verified by detecting the samples of previous external quality evaluation(EQA),compared with the comparative kits and recovery test.The lower limit of detection(LLD) was calculated by the results of blank samples.The cut-off value was verified according to the C28-A3C document approved by CLSI and CNAS-CL39:Guidance on the Application of Accreditation Criteria for the Medical Laboratory Quality and Competence in the Field of Clinical Qualitative Immunology respectively.The improved Doumas method was used to verify the range of linearity.Results The measured intra-assay and inter-assay coefficients of variation were lower than those announced by the manufacturer or the calculated values according to the EP15-A2 document.The coincidence rates for negative and positive EQA samples between detected and expected values were 98.4％ (63/64) and 100％ (20/20) respectively.The total coincidence rate was 98.8％ (83/84).The coincidence rate for negative and positive samples between the results from candidate and comparative kits were 91.2％ (52/57) and 87.0％ (40/46) respectively.The total coincidence rate was 89.3％ (92/103) and the Kappa value was 0.783 (P =0.062),which implied excellent consistency between the two kits.The mean recovery rate was 99.65％.The measured LLD was 0.5 IU/mL which was lower than 1 IU/mL as claimed by the manufacturer.The measured cut-off value according to the CNAS-CL39 document was 18.51 IU/mL,which was close to 20 IU/mL announced by the manufacturer.Based on the C28-A3C method,the cut-off value could be approved.The linear regression equation was Y =0.978 8X-3.125 4,r2 =0.996 1.There was no statistical difference between the intercept (-3.125 4) and 0 (t =-0.772,P =0.483).The range of linearity was from 1.6 to 212.5 IU/mL,which was consistent with the values declared by the manufacturer.All the verifications of the five performances above-mentioned could be passed.Conclusion The precision,accuracy,LLD,cut-off value and range of linearity of the candidate quantitative ELISA kit for anti-dsDNA antibody were consistent with the statement of the manufacturer,which indicated the performance of the kits may meet the requirements of clinic diagnosis and treatment.A series of methods used in this study provided a simple protocol for verifying the performance of quantitative ELISA kits.
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Objective To evaluate the diagnostic value of anti-nucleosome antibody for juvenile systemic lupus erythematosus (JSLE).Methods Fifty-four patients with JSLE,28 patients with non-JSLE and 26 healthy children were chosen in this study.antinuclear antibody(ANA),anti-nucleosome antibody (AnuA),anti-dsDNA antibody,anti-histone antibody (AHA) and anti-Sm antibody were detected by ELISA or western-blot method.The relevant clinical data were collected and analyzed.Results For diagnosis of JSLE,the sensitivity and specificity of AnuA was 77.78％ and 96.30％.The sensitivity of AnuA combined with ANA,anti-dsDNA and antiSm was higher than that of single detection.AnuA usually associated with fever,oral/nasal pharyngeal ulcer,lung damage,lymphocyte absolute value,urine protein and C3 level.Conclusion AnuA can be used as a serum marker for JSLE diagnosis.The detection of AnuA combined with anti-dsDNA and anti-Sm should be more helpful for diagnosis of JSLE.
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Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of diverse autoantibodies with various systemic organ involvements. In patients with SLE, autoantibodies, such as antinuclear antibody (ANA) and anti-dsDNA antibody, play an important role not only in diagnosing the disease, but also representing the pathogenesis of the disease. ANA is the main screening tool in diagnosis and serum complement levels and anti-dsDNA antibody level are closely related to the disease activities. Nevertheless, exceptionally, some patients represent with negative ANA and/or anti-dsDNA antibody leading to difficulties in diagnosing the disease. Here, we report a case of 37-year old female SLE patient with negative ANA, negative anti-dsDNA antibody, and positive anti-Ro/SSA antibody, which manifested with nephrotic syndrome.
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Female , Humans , Antibodies, Antinuclear , Autoantibodies , Autoimmune Diseases , Complement System Proteins , Glomerulonephritis, Membranous , Lupus Erythematosus, Systemic , Mass Screening , Nephrotic SyndromeABSTRACT
ObjectiveTo investigate the effect of arsenic trioxide (ATO) on anti-dsDNA antibody and the expression of DNA methyltransferase 1-mRNA and CD11a-mRNA in lupus MRL/lpr mice.Methods MRL/lpr mice were divided into three groups:the ATO group,the sodium chloride(NS) group,and the cyclophosphamide(CTX) group.The control group consisted of 20 syngeneic normal C57/BL mice,which were subdivided into the ATO group and the NS group.After two-month treatment,all mice were sacrificed.Blood routine test was conducted by SYSMEX KX21.The anti-dsDNA antibody in the serum were detected by ELISA.The expression of DNMT1 and CD11a was determined by RT-PCR.ANOVA and paired t test were used for statistical analysis.Results① The serum level of anti-dsDNA antibody(0.89±0.07) and the gray scale value of CD11a-mRNA(0.43±0.25) in the ATO group were much lower than those in the NS group of MRL/lpr mice(1.77±0.28,P＜0.01; 0.99±0.31,P＜0.05),while the gray scale value of DNMTI-mRNA (0.32±0.30) was significantly higher than that in the NS group(0.16±0.26,P＜0.05 ).② The serum levels of anti-dsDNA antibody was low in both the ATO group and the CTX group (0.90±0.07,0.66±0.22),and it was higher in the ATO group than that in the CTX group (P＜0.05).The gray scale value of DNMT1 mRNA in the ATO group (0.32±0.30) was higher than that in the CTX group (0.16±0.18,P＜0.05) in MRL/lpr mice,and the gray scale value of CD11a mRNA in the ATO group(0.43±0.25) was lower than that in the CTX group (0.86±0.31,P＜0.05) in MRL/lpr mice.③ There was no difference in above parameters between the ATO-group and NS group in C57/BL mice(P＞0.05).ConclusionArsenic trioxide can reduce the serum level of auto-antibody and reverse low methylation.But it has no effect on normal mice.
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In order to study the role of the bone marrow-derived mesenchymal stem cells(MSCs)transplanted with or without bone marrow(BM)in the treatment of lupus mice and the effect of MSCs in the onset of systemic lupus erythematosus(SLE).Method Twenty 12-week-old female MRL/lpr mice were randomly divided into four groups,including simple bone marrow transplantation group(SG,BM 1×107),united group-1(UG1,BM 1×107+MSCs 1×104),united group-2(UG2,BM 1×107+MSCs 1×106),the positive control group(PG,no transplantation).BALB/c mice were used as the negative control group(NG,no transplantation).MSCs which were amplified from the bone marrow of male BALB/c mice in vitro were transplanted into the female MRL/lpr mice with or without BM.One month later Y chromosome was detected to confirm if the transplantation was successful or not.The change of weight, white blood cells,urine protein,anti-dsDNA antibody,the pathology and immunofluorescence of renal were observed to evaluate the therapeutic efficacy.Results Y chromosome was detected in all transplanted female mice.Compared with PG,urine protein concentration in SG,UG1 and UG2 significantly decreased 30 days after transplantation(P<0.05).40 days after transplantation,the rite of anti-dsDNA antibodies in sG(0.91±0.27)was still higher than NG,which OD value wag 0.47 s0.10(P<0.05),but there was no statistical difference among UG1(0.76±0.28),uG2(0.73±0.10)and NG(P>0.05).However,50 days after transplantation,there was no marked difference of the tite of anti-dsDNA antibodies in SG(0.55±0.15),UG1(0.57±0.14)and UC2(0.58±0.05)compared with NG(P>0.05).After transplantation there was no vasculitis.no inflammatory cell infiltration in matrix and no obvious intercapillary cells proliferation in the kidney.The immunofluoroscence became negative or weakly positive.Conclusion MSCs transplantation with or without BM Can both improve the pathogenetic condition of MRL/lpr mice.MSCs can accelerate the clearance of anti-dsDNA antibody and promote the restoration of injured organs.We presume that MSCs are important immunological regulation cells in SLE.
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BACKGROUND: Detection of antibodies to extractable nuclear antigens (ENAs) and dsDNA is needed for the diagnosis of and predicting prognosis in systemic autoimmune diseases. Recently introduced line immunoassay (LIA) has the advantage of detecting several autoantibodies simultaneously, and we evaluated its usefulness in the diagnosis of autoimmune diseases in comparison with enzyme-linked immunosorbent assay (ELISA). METHODS: Samples were collected from 437 patients referred by rheumatologists. FANA (fluorescent antinuclear antibody) test and LIA for the detection of 13 different autoantibodies, including 6 ENAs and dsDNA were performed. LIA-positive samples for ENA or dsDNA antibodies were further tested with ELISA. Final diagnosis was made by rheumatologists according to the diagnostic criteria. Agreement of results between LIA and ELISA was analyzed in 53 selected patients with systemic autoimmune diseases. RESULTS: The LIA detected antibodies to ENA and dsDNA in 118 and 22 patients, respectively, and ELISA detected 70.3% (83/118) and 45.5% (10/22) of LIA positive samples. Especially, 60.2% (71/118) of patients with positive ENA antibody on LIA was diagnosed as systemic autoimmune diseases. Patients having strong FANA titer and homogenous/speckled pattern showed higher prevalence of autoantibodies, but a small proportion of FANA negative patients also showed positive reactivity (LIA 10.8%, ELISA 5.2%). LIA showed a good agreement with ELISA for the anti-ENA antibodies (> or =80%), and a lower agreement for the anti-dsDNA antibody (67.9%). CONCLUSIONS: LIA detecting several autoantibodies simultaneously might replace ELISA for anti-ENA antibodies, but not for anti-dsDNA antibodies. When LIA is performed considering clinical manifestations and FANA, it could contribute to the diagnosis of systemic autoimmune disease.
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Female , Humans , Male , Middle Aged , Antibodies, Antinuclear/analysis , Antigens, Nuclear/immunology , Autoimmune Diseases/diagnosis , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Immunoassay , Reagent Kits, Diagnostic , Reproducibility of ResultsABSTRACT
Objective To investigate the association between systemic lupus erythematosus(SLE) and polymorphism of cytotoxic T-lymphocyte antigen 4 gene(CTLA-4) and the effect on ANA and anti-dsDNA antibody.Methods The A/G substitution was investigated in the exon 1 position 49 in 92 SLE patients and 60 controls by PCR restriction fragment length polymorphism(PCR-RFLP).ANA and anti-dsDNA antibody were examined by indirect fluorescence immunoassay.Results SLE patients had significantly higher frequencies of the CTLA-4+49GG allele than controls(P
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BACKGROUND: In patients with systemic lupus erythematosus (SLE), serum factors play a role in the apoptosis and necrosis of neutrophils. We intended to verify that autoantibodies including anti-dsDNA antibody are one of the factors. We also investigated the potential usefulness of simultaneous flow cytometric measurement of cytotoxicity and autoantibody binding to neutrophils in SLE sera for evaluation of disease activity. METHODS: A total of 228 sera from 48 patients with lupus nephritis (LN), 19 patients with SLE without LN, 35 patients with rheumatoid arthritis (RA) and 40 healthy males were studied. Whole blood from healthy males mixed with test sera was incubated. Autoantibody binding, apoptosis and necrosis of neutrophils were measured by flow cytometry using IgG-FITC and 7-aminoactinomycin D after a 20-hour incubation period or after adjustments of incubation conditions. The results were expressed as the test/healthy control ratio of measured values, and the correlations between these results and anti-dsDNA antibody levels were investigated. RESULTS: IgG mean fluorescence intensity (MFI) ratio was 1.0+/-0.3, 1.6+/-1.6, 2.0+/-1.5 and 4.8+/-7.5 in the healthy, RA, SLE and LN group, respectively, and showed a significant increase in the LN group when compared with the healthy group (N=20 in each group, P<0.05). Apoptosis & necrosis ratio was 1.0+/-0.2, 1.0+/-0.5, 1.6+/-1.0 and 2.6+/-1.9 in each of the above 4 groups, and showed a significant increase in the LN group when compared with the healthy (P<0.005) and RA group (P< 0.01). By immunofluorescence microscopy, increased nuclear reactivities on neutrophils by autoantibody binding were observed in 12 (60%) of 20 LN sera. All three correlations between anti-dsDNA antibody level, apoptosis & necrosis ratio and IgG MFI ratio were significant (P<0.0005). Preincubation with DNA extracts decreased both IgG MFI ratio and apoptosis & necrosis ratio significantly (N=25, P<0.05). CONCLUSIONS: Our findings confirm the previous reports of increased neutrophil apoptosis in the peripheral blood of patients with SLE. This study indicates that anti-dsDNA antibody or other antinuclear antibodies in sera are associated with an active increase in the apoptosis and necrosis of neutrophils as well as simple binding to neutrophils. This may suggest that autoantibodies increase the exposure of autoantigen DNA and exacerbate autoimmunity in the pathogenesis. Although further studies are needed, we suggest that measuring the cytotoxicity and autoantibody binding to neutrophils in SLE serum simultaneously by flow cytometry should be a useful test for evaluation of disease activity.
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Humans , Male , Antibodies, Antinuclear , Apoptosis , Arthritis, Rheumatoid , Autoantibodies , Autoimmunity , DNA , Flow Cytometry , Fluorescence , Immunoglobulin G , Lupus Erythematosus, Systemic , Lupus Nephritis , Microscopy, Fluorescence , Necrosis , NeutrophilsABSTRACT
Objective To investigate the association of anti-C1q antibody with disease activity and lupus nephritis in patients with systemic lupus erythematosus(SLE). Methods The serum Level of anti-C1q antibody was determined in 35 patients with SLE by ELISA. The correlation between anti-C1q level and other disease activity parameters, such as SLEDAI score, anti-dsDNA antibody, C3, C4 and CH50 levels, was analyzed. Results Anti-C1q antibody was positive in 51.4% of patients with SLE, the positive rates in patients with active nephritis and without one was 77.8%(14/18) and 42.3%(22/52), respectively, and that was significantly higher in the former than in the later(P
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Objective To develop an ELISA for detection of anti-dsDNA antibody by using plasmid DNA as antigen.Methods DNA in plasmid pBV220 for prokaryotic expression vector was purified by base-cleavage. The microtiterplates were pretreated by poly-L-lysine and coated by the plasmid DNA in a dilution of 1∶50 as antigen. An ELISA method for detection of serum anti-dsDNA antibodies was developed with HRP-SPA as enzyme-labeled marker.The IIF using crithidia lucilia as substrate was performed simultaneously for comparison. The serum samples from 64 patients with SLE, 8 with MCTD and 17 with RA were detected. Results The concentration of DNA was 1 54 g/L by UV spectrophotometer at wavelength of 260 nm. The positive percentage of ELISA for anti-dsDNA was higher than that of IIF. By comparison with IIF the positive percentages in SLE, MCTD and RA groups were 23 4% vs 17 2%, 12 5% vs 12 5% and 11 8% vs 5 9%, respectively, and the coincident rates between the 2 methods were 93 8%, 100% and 94 1% respectively. The sensitivity and specificity of the developed ELISA for detection of anti-dsDNA were 100% and 93 4% when IIF was as gold standard.Conclusion The ELISA by using plasmid DNA as antigen to detect anti-dsDNA has fine precision, sensitivity and specificity. Its positive rate is higher than that of IIF thus it will contribute to monitor the activities for SLE patients′ condition.
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Objective:To study the relationship between the dose of active DNA and the induction of SLE-like syndrome.Methods: DNA from extracted ConA-activated spleen lymphocytes and immunized syngenic mice with different quantities of active DNA, the anti-dsDNA antibodies and anti-histone antibodies as well as the antibody subclass were detected by ELISA.The patterns of antinuclear antibodies and immune complexes in glomeruli were observed by immunofluorescent-stain. Results: 10 pig active DNA could induce all the animal to produce anti-dsDNA and anti-histone antibodies,and the induced autoann'bodies were mainly IgGl type.Only 25%animals produced autoantibodies immunized by 5 fjig active DNA. Conclusion:The minimum dose of active DNA to induce SLE-like syndrome was 10