异质性胞核核糖核蛋白A2在类风湿关节炎诊断中的临床意义

目的探讨异质性胞核核糖核蛋白A2(hnRNP A2)在类风湿关节炎(Rheumatoid Arthritis,RA)诊断中的临床意义,建立简便、快捷的ELISA检测方法,并与国外Anti-RA33 Ab试剂盒的结果对比,以作为验证国内试剂盒可用性的依据。方法以高纯度的具有抗原反应性的重组hnRNP A2蛋白作为抗原,对临床上包括137例RA在内的多种结缔组织病患者的血清中的相应抗体进行ELISA检测。应用国外Anti-RA33 Ab试剂盒ELISA法检测并对比检测结果。结果hnRNP A2在RA患者中的敏感性为37.23%,特异性为85.44%,抗hnRNP A2/RA33抗体在RA患者中阳性率均明显高于其他疾病组(P<0.05)。抗hnRNP A2/RA33抗体在早期RA患者中的阳性率为43.75%。与国外Anti-RA33 Ab试剂盒检测结果比较,在各组患者中检测抗RA33抗体的阳性率无明显差别(P>0.05),且总体符合率高达86.88%。另外,该抗体与年龄、病程、晨僵时间、血沉、C反应蛋白、关节功能、X线分期、类风湿因子(RF)、抗角蛋白抗体(AKA)、抗核周因子抗体(APF)及抗CCP抗体之间均无相关性(P>0.05)。结论我们初步纯化了RA33抗原,对hnRNP A2进行了基因的克隆、重组蛋白的表达和纯化,并以其为抗原,用ELISA方法检测抗hnRNP A2/RA33抗体是早期诊断RA的可靠方法。与国外Anti-RA33 Ab试剂盒的检测结果对比无明显差异,自制的抗RA33抗体试剂盒可以可靠地应用于临床检验。

Preparation of Recombinant Islet Cell Autoantigen 69 kD Fusion Protein

To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.

Autoantibodies against MMP-7 as a novel diagnostic biomarker in esophageal squamous cell carcinoma

AIM： To evaluate the diagnostic values of serum autoan tibodies against matrix metalloproteinase-7 （MMP-7） in patients with esophageal squamous cell carcinoma （ESCC）. METHODS： The MMP-7 cDNA was cloned from ESCC tissues, and MMP-7 was expressed and purified from a prokaryotic system. MMP-7 autoanUbodies were then measured in sera from 50 patients with primary ESCC and 58 risk-matched controls, using a reverse capture enzyme-linked immunosorbent assay （ELISA） in which autoantibodies to MMP-7 bound to the purified MMP-7 proteins. In addition, MMP-7 autoantibody levels in sera from 38 gastric cancer patients and from control serum samples were also tested. RESULTS： The optimum conditions for recombinant MMP-7 protein expression were determined as 0.04 mmol/L Isopropyl-13-D-Thiogalactopyranoside （IPTG）induction at 37℃ for four hours. The levels of serum autoantibodies against MMP-7 were significantly higher in patients with ESCC than in the matched-control samples （OD450 = 1.69 ±0.08 vs OD450 = 1.55 ± 0.10, P 〈 0.001）. The area under the receiver operating character- istic （ROC） curve was 0.87. The sensitivity and specificity for detection of ESCC were 78.0% and 81.0%, respectively, when the OD450 value was greater than 1.65. Although the levels of autoantibodies against MMP-7 were also significantly higher in patients with gastric cancer compared to control samples （OD450 = 1.62± 0.06 vs OD450 = 1.55±0.10, P 〈 0.001）, the diagnostic accuracy was less significant than in ESCC patients. The area of ROC curve was 0.75, whereas the sensitivity and specificity were 60.5% and 71.7%, respectively, when the cut-off value of OD450 was set at 1.60. CONCLUSION： Serum autoantibody levels of MMP-7 may be a good diagnostic biomarker for esophageal squamous cell carcinoma,

Levels of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 in gastric cancer

AIM:To evaluate the levels of preoperative serum matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in gastric cancer.METHODS:One hundred gastric cancer patients who underwent gastrectomy were enrolled in this study.The serum concentrations of MMP-1 and TIMP-1 in these patients and in fifty healthy controls were determined using an enzyme-linked immunosorbent assay.RESULTS:Higher serum MMP-1 and TIMP-1 levels were observed in patients than in controls (P < 0.001).Serum MMP-1 and TIMP-1 levels were positively associated with morphological appearance,tumor size,depth of wall invasion,lymph node metastasis,liver metastasis,perineural invasion,and pathological stage.They were not significantly associated with age,gender,tumor location,or histological type.CONCLUSION:Increased MMP-1 and TIMP-1 were associated with gastric cancer.Although these markers are not good markers for diagnosis,these markers show in advanced gastric cancer.

Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

Usefulness of fecal lactoferrin in predicting and monitoring the clinical severity of infectious diarrhea

AIM:To explore the value of fecal lactoferrin in predicting and monitoring the clinical severity of infectious diarrhea.METHODS:Patients with acute infectious diarrhea ranging from 3 mo to 10 years in age were enrolled,and one to three stool samples from each subject were collected.Certain parameters,including white blood cells /differential count,C-reactive protein,fecal mucus,fecal pus cells,duration of fever,vomiting,diarrhea and severity(indicated by Clark and Vesikari scores),were recorded and analyzed.Fecal lactoferrin was determined by enzyme-linked immunosorbent assay and compared in different pathogen and disease activity.Generalized estimating equations(GEE) were also used for analysis.RESULTS:Data included 226 evaluations for 117 individuals across three different time points.Fecal lactoferrin was higher in patients with Salmonella(11.17 μg/g ± 2.73 μg/g) or Campylobacter(10.32 μg/g ± 2.94 μg/g) infections and lower in patients with rotavirus(2.82 μg/g ± 1.27 μg/g) or norovirus(3.16 μg/g ± 1.18 μg/g) infections.Concentrations of fecal lactoferrin were significantly elevated in patients with severe(11.32 μg/g ± 3.29 μg/g) or moderate(3.77 μg/g ± 2.08 μg/g) disease activity compared with subjects with mild(1.51 μg/g ± 1.36 μg/g) disease activity(P < 0.05).GEE analysis suggests that this marker could be used to monitor the severity and course of gastrointestinal infections and may provide information for disease management.CONCLUSION:Fecal lactoferrin increased during bacterial infection and with greater disease severity and may be a good marker for predicting and monitoring intestinal inflammation in children with infectious diarrhea.