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Objective To assess the interference by calcium dobesilatein 7 peroxidase-baseduric acid assays and to determine its clinical significance.Methods In the in vitro experiments, uric acid in pooled serum with final concentrations of calcium dobesilate additions (0, 2, 4, 8, 16, 32, and 64μg/ml) were measured by 7 peroxidase-based assays.Percent Bias (%) was calculated relative to the drug-free specimen.In the in vivo experiments, changes in serum uric acid and calcium dobesilate concentrations were observed before and after calcium dobesilate administration ( baseline, 0 h, 1 h, 2 h, 3 h, 4 h, 6 h ) involunteers.The interference in different assays was assessed compared with LC-IDMS/MS method. Calcium dobesilate levels in 40 specimens from those taking calcium dobesilate were measured by HPLC method.Of the 40 specimens, 10 were selected to analyse the levels of uric acid by both peroxidase and UV measurement method to assess the impact in clinical status.Results In the in vitro study, concentrations of uric acid measured by 7 peroxidase-based assays were reduced by -6.3%to -21.2%compared with drug-free serum, when theconcentration of calcium dobesilate was16μg/ml.In the in vivo study, comparedto UA levels at 0 h, the biasesof serum uric acid determined by peroxidase method after calcium dobesilate administration(1 h, 2 h, 3 h, 4 h, 6 h) were of -3.33%, -6.79%, -7.49%, -6.07%, -4.09%, respectively.The observed uric acid concentrations for 8 participants measured by enzymatic assays were inhibited by -3.75% to -6.89% at 0 hour and by -16.9% to-22.22% at 2 hours relative to the concentrations measured by the LC-IDMS/MS method. Conclusions Calcium dobesilate produced a clinically significant negative interference with uric acid in all peroxidase-based uric acid assays,which may result in false evaluation of uric acid level in clinical status.Significant differences in the degree of interference were observed among the assays.
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Objective To assess the consistency of four standardized cystatin C particle-enhanced turbidimetric assay (PETIA) and one particle-enhanced nephelometric immunoassay (PENIA) measurement systems Methods Performance verification test was conducted according to CLSI EP 15-A2 and EP9-A2. Fourty serum samples in comparative test were obtained from the remaining serum samples of outpatients in Peking Union Medical College Hospital in March 2013.Fourty serum samples were tested on Olympus AU5400 automatic biochemical analyzer ( four PETIA Cys C reagents:Shanghai Jingyuan Co ., Ltd, Beijing Leadman Biochemistry Co ., Ltd, Beijing Strong Biotechnologies , Maker Biotechnology in Sichuan , and labelled as A, B, C, D respectively) and PENIA N Latex Cys C measurement system on Siemens BNⅡ(labelled as E).Correlation analysis were performed among four PETIA methods one PENIA method Differences of each detection system were compared in the medical decision level 1,2,3,4 mg/L.The reference material ERM-DA471/IFCC was measured by five systems and bias ( percentage bias ) was calculate for each system.Results Results of systems A, B, C, D, E were 1.29(0.89-2.43), 1.30 (0.96-2.59), 1.22(0.90-2.44), 1.27(0.96-2.47), 1.14(0.82-2.05)mg/L.Chart shows bias among these five systems was small when Cys C concentration was less than 4mg/L.PETIA method A, B, C, D correlated with their mean value well , with the average deviation from their mean value ( percent deviation) at -0.017 -0.031 mg/L ( -3.1%-2.1%), and all were less than allowed bias from the biological variation (3.4%).The deviation of PETIA method A, B, C, D with their mean value in medical decision level at -0.176 -0.178 mg/L.Systems A, B, C, D correlated well with the result of PENIA method system E , and the mean deviation ( percent deviation ) was at 0.278 -0.326 mg/L ( 12.6%-18.5%) , and the deviation ( percent deviation ) in the medical decision level 0.055 -1.079 mg/L (5.51%-26.98%).Bias of PETIA method A, B, C, D Cys C system measuringERM -DA471/IFCC ranged from 0.22 to 0.39 mg/L ( 3.9%-7.0%) , which exceeded the allowable range of the reference material target value, and were larger than the allowable bias from biological variation (3.4%).Bias ( percent ) of PENIA method system E was -0.1 mg/L ( -1.7%) , within the allowable range of ERM-DA471/IFCC target value .Conclusions The consistency of four assesed PETIA Cys C reagents was relatively ideal, and improved markably after being traced to ERM-DA471/IFCC.Besides, the results of PETIA were higher than those of PENIA .Bias among these five systems was small when Cys C concentration was less than 4 mg/L, and the bias became larger in higher Cys C concentration.
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Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.
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Objective To detect the value of autoantibedy profile in primary biliary cirrhosis (PBC). Methods 96 serum samples of patients with primiary biliary cirrhosis, 100 serum samples of other antoimmune disease and 49 serum samples of healthy were tested for anti- M2, anti-3E(BPO), anti-Sp100,anti-PML,anti-gp210,anti-LKM-1 ,anti-LC-1 ,anti-SLA/LP by EUROLine. Results The positive of the anti-M2,anti-BPO, anti-Sp100, anti-PML and anti-gp210 for PBC was 76. 0%, 84.4%, 32. 3%, 28. 1% and 35.4% ,respectively. The positive of the anti-M2, anti-BPO, anti-Sp100, anti-PML and anti-gp210 for other autoimmune disease was 13.0% ,9. 0% ,3.0% ,2.0% and 1. 0%, respectively. The sensitivity of the anti-M2 for PBC was 76. 0% ,with specificity of 87. 0%. The sensitivity of the anti-BPO for PBC was 84. 4%, with specificity of 91.0% ;the sensitivity of the anti-Sp100 for PBC was 32. 3%, with specificity of 97.0%. The sensitivity of the anti-PML for PBC was 28. 1% ,with specificity of 98.0%. The sensitivity of the anti-gp210 for PBC was 35.4%, with specificity of 99. 0% . Anti-LKM-1, anti-LC-1, anti-SLA/LP positive patients with PBC were not detected;the incidence rate of liver function failure in anti-gp210 positive serum higher than anti-gp210 negative serum (χ2 = 11.17, P < 0. 01). Conclusions Multiple autoantibedies can be detected in the sera of PBC patients. The detection of autoantibody profile is useful for the diagnosis and differential diagnosis of PBC, and may he helpful for therapy and prognosis of PBC.
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Objective To investigate the association of TGF-β receptor typeⅡ(TβRⅡ)mRNA with lupus nephritis (LN) and disease activity by testing its expression levelin peripheral blood mononuclear cells (PBMCs).Methotis Forty-four patients with LN were included in this study.They were all had active LN.Twepty-eight LN patients were taking glueocorticoids and/or immunosuppressive agents and sixteen had never taken steroids or immunosuppressive agents.The expression levels of T13R H mRNA were semi-quantitativelydetermined by reverse transcription-polymerase chain reaction(RT-PCR).Resuits The expression levels of TβRⅡ mRNA in PBMCs from LN patients(1.7±1.0)were lower than those of non-lupus nephritis(4.0±3.1) and healthy subiects(4.1±2.5),(P<0.01).The difference of the expression levels between patients who took and had never taken glucocorticoids and/or immunosuppressive drugs was significantly statistically(P<0.05).The expression levels of TβRⅡ mRNA in PBMCs of patients with LN were correlated significantly with the systemic lupus erythematosus disease activity index (SLEDAI)scores(r-0.309.P<0.05),titers of anti-dsDNA antibody(r=-0.401,P<0.01)and serum complement C3 level(r=0.621,P<0.01).Conclusion This study suggests that TβRⅡ may be involved in the development of LN,and the TβRⅡ mRNA expression levels in PBMCs from patients with SLE are significantly correlated with LN activity.Glucocortieoids or immunosuppressive drugs can increase the expression levels of TβRⅡ mRNA and ameliorate renal damage.
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Objective To explore the relationship of Toll-like receptors (TLR)-3 Mrna expression level on peripheral blood monocytes (PBMCs) with disease activity of PBC in patients with primary biliary cirrhosis (PBC). Methods The expression level of TLR-3 Mrna on peripheral blood monocytes was tested in 55 PBC cases (33 cases in active phase, 22 cases in stable stage), 20 cases with hepatoma (as disease controls) and 24 healthy controls. By real-time quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) methods. Β-actin was selected as the internal control. △Ct=Ct (target gene)-Ct(internal reference gene) were used to measure the gene expression level. Results The mean TLR-3 Mrna expression of PBC in active stage was higher than that of stable stage, hepatoma (P<0.001) as well as normals.The difference was significant (P=0.011<0.05). The mean TLR-3 Mrna was not different than that of the stable stage (P=0.221>0.05) and normal controls (P=0.347>0.05). There was no difference between patients with stable PBC and normal controls (P=0.590>0.05). Conclusion The TLR-3 Mrna expression level of PBMCs is elevated in active PBC patients. It may correlate with the pathogenesis and disease activity of PBC.
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Objective To detect the expression of GITR mRNA in peripheral blood mononuclear cells (PBMCs) of patients with primary biliary cirrhosis (PBC) and its relationship with the disease activity of PBC.Methods Real time quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) was used to examine GITR mRNA expression on peripheral blood monocytes of 44 active PBC, 34 stable PBC, 30.hepa-toma cases and 30 healthy controls. Results The mean GITR mRNA expression of active PBC (△Ct=10.5±
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Objective To evaluate the clinical significance of combined measurement of antiperinuclear neutrophil cytoplasmic antibody(ANCA)and anti-saccharomyees cerevisia antibody(ASCA)for the diagnosis of inflammatory bowel disease(IBD)patients and difierentiation of Crohn's disease(CD)with ulcerative colitis(UC).Methods A total of 159 patients with IBD(97 UC,62 CD),167 patients with other non-IBD gastrointestinal conditions(NIBDC)and 25 healthy controls(HC)were recruited in our research.ASCA and ANCA were detected by enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence assay.respectively.Results The prevalence of ASCA-IsA or IgG in CD group,UC group,NIBDC and HC were43.5%,14.4%,29.3%and 0,respectively.The prevalence of ASCA-IgA or IgG in CD group were higher than those in other groups(X2=16.76 or 4.12,P＜0.01 or＜0.05).The prevalence of ANCA in CD group.UC group,NIBDC and HC were 8.1%,56.7%,4.8%and 0,respectively.The prevalence of AMA in UC group were much higher than those in other groups(X2=38.08 or 90.47,P＜0.01).The sensitivity specificity and positive predictive value(PPV)in ASCA+/ANCA-were 40.3%.93.8% and 80.6%,respectively,and in ANCA+/ASCA-were 48.5%,98.4% and 97.9%,respectively.Condusions ASCA or ANCA testing alone are not sensitive enoulgh for diagnosing CD and UC,but their combination asses are specific for differential diagnosis between CD and UC.Combined testing of ASCA-IgA with IgG can improve the sensitivity in screening CD patients.The ASCA positive pattern in Chinese CD group are correlated with surgery.
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Objective To study the relationship between the expression of eostimulating factor B7-H4 in patients with primary biliary cirrhosis (PBC) and the pathogenesis of PBC. Methods The expression of B7-H4 mRNA on peripheral blood mononuclear cell (PBMC) of 65 patients with PBC was tested by real-time PCR. Serum levels of IL-2 were assayed by ELISA. CD4+, CD8+ T lymphocytes expression level and B7-H4 expression rate before and after activation were measured by three-color flow cytometry (FCM). Re-sults (1) Expression of B7-H4 mRNA and BT-H4 percentage in PBC group were significantly lower than that in none-PBC group and healthy controls(P<0.01);(2)After 72 h activation, the percentage of CD4+, CD8+, and CD4+ CD8+T lymphoeytes and serum levels of IL-2 decreased (P<0.05), and the percentage of CD4+, CD4+ CD8+T lymphocytes and serum levels of IL-2 were significantly higher than that of none-PBC group and healthy controls(P<0.01);(3)Levels of alanine aminotransferase(ALT), aspartate amin-otransferase(AST), alkaline phosphatase(ALP) and γ-glutamyl transpeptidase(GGT) of patients with posi-tive anti-mitochondrial antibody(AMA)-M2 rose. There was no significant difference of B7-H4 expressions on T cells between patients either with or without AMA-M2 antibody. Conclusion The costimulating factor B7-H4 can express on T lymphocytes which is activated by phytohemagglatinin(PHA) aad plays a negative role on T cells responses.
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Objective To investigate the relationship between the PTPN22 gene polymorphism and rheumatoid arthritis(RA).Methods Real time fluorescent quantitation PCR was used to detect the 1123G＞C polymorphism of the PTPN22 gene from 200 RA patients,100 others rheumatic diseases and 200 the normal controls.The results were analyzed by SPSS 11.0 software.Results The CC genotype frequencies of RA patients.others rheumatic diseases and the normal controls were 0.120,0.020 and 0.015 respectively.There was a significant difiefence between BA patients and others rheumatic diseases (X=18.708.P＜0.01).1'here Wag a significant difference between RA patients and the normal controls(X2=24.337,P＜0.01).There was not statistically significant between others rheumatic diseases and the normal controls(X2=1.066,P＞0.05).The C allele frequency of RA patients,others rheumatic diseases and the normal controls were 0.360.0.190 and 0.215 respectively.The results were significant difference.Conclusion The PTPN22 gene could be one of predisposing genes and the therapeutic target genes with RA patients.
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Objective To clone and construct the recombinant plasmid containing Jo-1 of HepG2 cells,then purify the protein and identify the immunoreactivity of the recombinant protein.and establish the enzyme linked immunosorbent assay(ELSA)to detect Jo-1 autoantigen correlative antibodies in diagnosis of polymyositis/dermatomyositis.Methods The constructed plasmid was transformed into E.coli.DH5α and BL21(DE3).This fusion protein was purified by Ni-NTA chromatography and its immunnoreactivity was identified by SDS-PAGE and Western blot.ELISA with the fusion protein was established to detect the Jo-1 autoantigen correlative antibodies in sernm samples of 75 patient with PM/DM,30 patients with SLE.30 patients with RA,10 patients with SS and 30 normal controls.Results The sequence of Jo-1 autoantigen gene Was the same as the sequence reported on the literatures.SDS-PAGE gel analysis showed the molecular weisat of fusion protein was approximately 55 000 Da. Western blotting analysis showed that the fusion protein had the same immunoreactivity as human Jo-1 autoantigen.The results of ELISA indicated that the positive rate of anti-Jo-1 antibody was 28%.but the antibody was negative in other controls.There was significant difierence of positivity of the autoantibody between PM/DM and disease controls or normal controls (x2=31.84,P＜0.01).Conclusions The plasmid containing Jo-1 is successfully cloned into E.coli.DH5α and BL21 (DE3).EUSA analysis shows its good antigenicity and specificity.
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Objectlve To detect the expression of CXCR3 mRNA in peripheral blood mononuclear cells (PBMC) in patients with primary biliary cirrhosis (PBC) ,and explore its relationship with activity of PBC.Methods Reverse transcription-real time quantitative polymerase chain reaction (FQ-RT-PCR) was used to examine CXCR3 mRNA expression in peripheral blood monocytes of 29 cases of PBC in active stage. 30 cases in stable stage,20 cases in CTD,and 30 healthy controls.Results The mean level of CXCR3 mRNA expression of PBc inactive stage (△Ct=5.41±2.69) Was higher than that of stable stage (△Ct=7.77±2.74,t=3.39,P＜0.01),CTD(△Ct=7.24±2.75,t=2.53,P＜0.01),and healthy controls (△Ct =7.16±2.76,t=2.45,P＜0.01).There was no significant difference of expression levels of CXCR3 mRNA among stable stage PBC patients,CTD diseases patients and healthy controls (P＞0.05).Conclusion This study indicated that the CXCR3 mRNA expression levels of PBMC is significantly elevated in patients with active PBC.and it could be implicated in pathogenesis and activity of disease.
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Objective To establish and apply the protein chip to detect eleven autoantibodies profile, and evaluate the authenticity and reliability with ANAs protein chip in clinical autoantibodies profile detection.Methods By comparing the results of IIF and ELISA , validation the sensitivity and specificity of ANAs protein chip in clinical autoantibodies profile detection. The autoantibodies detected were anti-SSA-52,anti-SSA-60,anti-SSB,anti-Sm,anti-RNP,anti-Scl-70,anti-Jo-1,anti-dsDNA,anti-rRNP,anti-centromere antibodies and antinuclear antibodies (ANA). To each autoantibody, we have selected 70 positive and 294 negative samples except the 32 rare samples that contain anti-Jo-1 antibody.Results The sensitivity to all the autoantibodies was 100% except anti-SSA52 and anti-SSB antibodies was 95.7%and 98.6% respectively. The specificity to all the autoanbodies was 100% except anti-SSB, anti-RNP-68, anti-Scl-70, anti-dsDNA, anti-CENP-B and ANA was 98.0%, 98.0%, 99.7%, 99.7%, 99.7% and 98.3% respectively. Conclusions To all the eleven antinuclear autoantibodies , the sensitivity is all above 95.0% and specificity is all above 98.0%, which indicate that there is high concordances between the ANAs protein chip and the methods used in clinical screening and confirmation,and it could meet the requirement of clinical autoantibodies profile detection. The protein chip method is fast, easy for detection with the characteristic of high-throughput,high sensitivity and specificity,it is hence recommended to apply ANAs protein chip to detect autoantibodies profile in clinical detection.
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Objectives To investigate the ratios of peripheral blood CD4+CD8+ and CD4+CD25+ regulative T cells, and explore the association with hepatic damnification and anti-AMA-M2 antibodies.Methods The percentage of CD4+CD8+T cells and CD4+CD25+T cells in peripheral blood from patients with primary biliary cirrhosis(PBC) (n=27)、26 patients with other hepatic desease、30 normal individuals were measured by flowcytometry.Results Patients with PBC had statistically higher levels of CD4+CD25+T cells than the patients with other hepatic disease (P
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Objective: To investigate the ratios of peripheral blood CD4+CD25+,CD4+CD8+ regulative T cells of systemic lupus erythematosus(SLE) patients, and explore the association with disease active stage,nephropathy,serum anti-ds-DNA antibody,and both IgG and C3 levels. Methods: The percentage of CD4+CD25+T cells and CD4+CD8+T cells of peripheral blood from patients with systemic lupus erythematosus(SLE)(30 females and 7 males),30 rheumatism controls and 30 normal individuals were measured by flowcytometry. Results: Patients with active disease had statisitically lower levels of CD4+CD25+T cells than did normal controls(P