Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbant assay:optimization,standardization and diagnostic criteria

Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.

Location-based prediction model for Crohn’s disease regarding a novel serological marker,anti-chitinase 3-like 1 autoantibodies

BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.

新霉素ELISA检测方法的建立

目的:比较直接和间接竞争酶联免疫法(enzyme linked immunosorbent assay,ELISA)的优缺点,建立新霉素残留ELISA检测方法。方法:利用自制的新霉素多克隆抗体,采用直接竞争和间接竞争ELISA方法检测新霉素残留,并比较两种方法的优缺点。结果:新霉素抗血清和庆大霉素的交叉反应率为2.04%,和卡那霉素的交叉反应率为0.02%,和氨苄青霉素、红霉素、四环素的交叉反应率均小于0.01%。初步测试新霉素间接竞争ELISA法的准确性和回收率。板内误差小于4%,板间误差小于11%,回收率为135.5%～191.3%。直接竞争和间接竞争ELISA方法的检测极限分别为28.58ng/mL和51.74ng/mL,达到了国家对新霉素规定的500μg/kg MRL检测限。结论:建立了直接竞争和间接ELISA吸附检测方法,条件优化更成功的间接竞争ELISA可用于开发新霉素检测试剂盒。

重症大疱性类天疱疮患者血清抗体变化规律与病情相关性的研究

目的：研究重症大疱性类天疱疮（ bullous pemphigoid ， BP ）患者血清中抗体BP180NC16a的酶联免疫吸附试验（ELISA）指数变化情况，观察其与病情变化的相关性，并分析用于病情监测和指导治疗的临床意义。方法对12例皮损面积＞50％的重症大疱性类天疱疮患者血清抗体BP180 NC16 a水平在不同时期进行监测及评分，并分析之间的关系。结果12例患者，平均年龄65岁，皮损面积均大于全身体表面积的50％以上。皮疹主要表现为疱壁紧张的大疱、水疱，部分有口腔黏膜损害。患者皮损面积和病情评分与血清抗体BP180NC16a-ELISA指数具有显著性关联（P＜0．05），患者疾病活动期和临床缓解期抗体BP180NC16a-ELISA指数几乎与病情呈平行变化，并且该指数可以预测病情，从而指导治疗。结论重症大疱性类天疱疮多为老年患者，病情危重，在发病早期不易诊断，因而延误治疗导致死亡。血清中抗体BP180 NC16 a-ELISA 指数可反映疾病的活动程度，用于病情监测，为治疗时根据个体差异选用适量的糖皮质激素快速控制病情提供了有利的实验室证据。

食品中丙烯酰胺检测方法的研究进展

丙烯酰胺是在食品高温加工过程中产生的小分子有机化合物,具有致癌性。从样品提取、衍生化、净化、富集等前处理过程以及对检测器的选择出发,总结国内外近年来用于检测丙烯酰胺的方法,如从传统的气相色谱、液相色谱及其联用技术,到新兴开发的分子印迹技术、酶联免疫吸附和生物传感器等新检测技术。并根据其适用范围和操作条件,对各分析方法的优点和不足进行讲述,最后对将来丙烯酰胺检测方法发展新思路提供策略和依据。

乙型肝炎表面抗原不同检测方法的优势比较分析

目的：比较化学发光微粒子免疫分析法（CMIA）和酶联免疫吸附法（ELISA）用于乙型肝炎（乙肝）表面抗原检测的优势效果。方法选取2011年3月～2013年4月采用ELISA法与CMIA法对386例患者进行了乙肝表面抗原检测的对比研究。结果 CMIA法检测乙肝表面抗原的阳性率为51.3%，ELISA法阳性率为43.5%，CMIA法阳性率明显更高，与ELISA法比较差异有统计学意义（P〈0.05）；CMIA法的灵敏度与特异性均明显高于ELISA法（P〈0.05）。结论与ELISA法比较，CMIA法对乙肝表面抗原检测在阳性率、灵敏度以及特异性等方面均存在有比较明显的优势，故建议将CMIA法作为临床检测乙肝表面抗原的首选方法而推广应用。

鼻息肉组织中T淋巴细胞功能的研究

鼻息肉是多因素引发和介导的一种炎症性疾病（[1]）,多种炎性细胞浸润是其主要病理学特征（[2]）。我们前期的研究发现,鼻息肉组织中可见大量树突状细胞浸润[3],树突状细胞将抗原递呈给T淋巴细胞后,两者通过一系列的相互作用,介导免疫反应,而T细胞的分化失衡在鼻息肉发病过程中起到关键性作用（[4]）。然而,鼻息肉组织中T淋巴细胞的功能状态目前尚不明确。

单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展

近20年来,人们制备了许多小麦种子贮藏蛋白的单克隆抗体(Monoclonal Antibody,McAb),一方面作为有效工具研究胚乳贮藏蛋白(主要是麦谷蛋白聚集体、特定的谷蛋白亚基及醇溶蛋白)的结构与功能关系;另一方面用作诊断试剂(diagnostic tools),为筛选具有合适加工品质的小麦品种提供依据。本文综述了国内外单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展,并展望其应用前景。

三甲氧苄氨嘧啶单克隆抗体制备以及酶联免疫试剂盒的研究

[目的]探讨系统地检测水质中三甲氧苄氨嘧啶残留量的方法。[方法]通过三甲氧苄氨嘧啶与马来酸酐反应，得到三甲氧苄氨嘧啶半抗原，再通过免疫动物得到抗三甲氧苄氨嘧啶单克隆抗体，并将其应用于能够检测水质中三甲氧苄氨嘧啶残留量的ELISA试剂盒。[结果]试验表明，该试剂盒对水质中三甲氧苄氨嘧啶的检测限为2．34μg／kg，IC50（50％抑制浓度）为4．8μg／L，回收率为60．5％～79．7％，试剂盒的标准曲线范围为0～80μg／L，批内、批间的相对标准偏差均小于10％，三甲氧苄氨嘧啶单克隆抗体与二甲氧苄氨嘧啶的交叉反应率小于1％，4℃下能够保存12个月，稳定性较好。[结论]研究可为监管三甲氧苄氨嘧啶的滥用提供参考。

Detection of <i>Chlamydia trachomatis</i>and <i>Neisseria gonorrhoeae</i>in Egyptian Women Suffering from Infertility

Chlamydial and gonococcal infections are recognized as two of the major causes of sexually transmissible human bacterial infection which may lead to infertility. In this cross sectional study, we aimed to determine the prevalence of Neisseria gonorrhoeae, Chlamydia trachomatis among Egyptian women using different microbiological methods. One hundred and fifty cervical swabs were collected, of which 100 were from infertile women. Culture and ELISA technique were used for screening of Neisseria gonorrhoeae and Chlamydia trachomatis individually. In addition, PCR was used for all examined samples. For C. trachomatis, 3 cases were positive for antigen detection by ELISA. Moreover, in obtained results of PCR, DNA was detected in 4 samples, and three of them from infertile group. So based on PCR results, the sensitivity and specificity of ELISA were 75% and 100% respectively. Furthermore, 3 samples were positive for gonococcal infections by PCR, and two of them were taken from infertile women. Positive results of two samples were verified by culture. The estimated sensitivity and specificity of culture method were 66.7% and 100% respectively. Results of this study indicate that PCR is a valuable method for detection of gonococcal and chlamydial infection and it is suitable for the confirmation of ELISA results for C. trachomatis diagnosis. Culture method is less sensitive than PCR for detection of N. gonorrhoeae. The prevalence of such infections is higher among infertile women.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone.Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up. PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomicDNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgeneexpressing cells was 65 ± 10 ng / L per 106 cells,420 ± 45 ng/L per 106 cells in sense group and 485± 30 ng/L per 106 cells in the negative control group, (P＜0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S. C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.

Serum vascular endothelial growth factor is a potential biomarker of metastatic recurrence after curative resection of hepatocellular carcinoma

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the mostcommon malignancies in China.To date,surgery is stillthe best solution to it.However,metastatic recurrencesafter curative hepatic resections are very common.Tang etal have reported that recurrence rate within 5 years

Effect of vitamin E succinate on expression of TGF-β_1, c-Jun and JNK1 in human gastric cancer SGC-7901 cells

INTRODUCTIONVitamin E succinate (RRR-α-Tocopheryl Succinate,VES), a derivative of natural vitamin E, is acompound esterified by succinic acid and 6-hydroxyl-α-tocopheryl.

Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys

INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].

Level of circulating PD-L1 expression in patients with advanced gastric cancer and its clinical implications

Objective：The programmed cell death-1 receptor/programmed cell death-1 ligand （PD-1/PD-L1） pathway plays a crucial role in tumor evasion from host immunity.This study was designed to evaluate the association between circulating PD-L1 expression and prognosis in patients with advanced gastric cancer.Methods：Totally 80 advanced gastric cancer patients and 40 health controls from Beijing Cancer Hospital were enrolled in the present study.Circulating PD-L1 expression was tested by enzymelinked immunosorbent assay （ELISA）.The associations between the expression level of PD-L1 and clinicopathological features and prognosis were analyzed statistically.Results：Expression of PD-L1 in advanced gastric cancer patients was significandy up-regulated compared with health people （P=0.006）.The expression of PD-L1 was significantly correlated with differentiation and lymph node metastasis （P=0.026 and P=0.041,respectively）.Although we didn＇t find significant difference in all advanced gastric cancer patients with different PD-L1 expression,the adenocarcinoma patients with higher up-regulated PD-L1 expression had much better prognosis than low expression patients （65.6％ vs.44.7％,P=0.028）.Conclusions：PD-L1 was elevated in advance gastric cancer patients and may play an important role in tumor immune evasion and patients prognosis.

Changes of p53 protein blood level in esophageal cancer patients and normal subjects from a high incidence area in Henan,China

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Serum Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Level in Patients with Colorectal Carcinoma and Clinical Significance

Circulating vascular endothelial growth factor-C （VEGF-C） and vascular endothelial growth factor （VEGF） levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay （ELISA）. Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76 % with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72 % sensitivity and 74 % specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3 %, the negative predictive value was 94.6 %, and accuracy was 93.7 %. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.

Angiogenesis-related cytokines in serum of proliferative diabetic retinopathy patients before and after vitrectomy

AIM: To evaluate serum concentrations of angiogenesis-related cytokines in proliferative diabetic retinopathy (PDR) before and after vitrectomy. METHODS: Serum samples were collected from 30 PDR patients with varying severity before and after vitrectomy. Serum concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), interleukin-8 (IL-8) and interferon-inducible protein-10 (IP-10) were determined by enzyme-linked immunosorbent assays(ELISA). RESULTS: Serum concentrations of VEGF, PEDF, IL-8 and IP-10 were significantly higher in PDR patients than that in controls, respectively (P<0.05). VEGF concentration decreased significantly in postoperative samples than that in preoperative samples (P<0.05). The concentrations of PEDF, IL-8 and IP-10 did not exhibit significant changes after vitrectomy. CONCLUSION: Elevated cytokines levels in serum may be diagnostically useful in PDR. Angiogenesis-related cytokines play important roles in the development of PDR, and would instruct the risk assessment of pathogenetic condition in PDR patients.

Cartilage oligomeric matrix protein: A novel non-invasive marker for assessing cirrhosis and risk of hepatocellular carcinoma

AIM: To assess serum cartilage oligomeric matrix protein(COMP) as a marker of cirrhosis and risk of progression to hepatocellular carcinoma(HCC). METHODS: A COMP enzyme-linked immunosorbentassay was used to test 187 patients with chronic liver diseases at the time point of first evaluation. The selected patients included 72 with chronic hepatitis B infection, 75 with chronic hepatitis C infection, 22 with primary biliary cirrhosis, 7 with autoimmune hepatitis type 1, and 11 with alcoholic liver disease. Demographic, biochemical, histological and clinical characteristics of the patients were recorded at the first evaluation. One hundred and forty-seven patients were followed for a median [interquartile range(IQR)] duration of 96.5(102) mo. The clinical, biochemical and histological data, as well as the development of cirrhosis, HCC according to internationally accepted criteria and in case of death, a liver-related cause during the follow-up period, were recorded at the electronic database of our clinic. COMP determination was also performed in 43 healthy individuals who served as the control study group.RESULTS: COMP positivity(> 15 U/L) was detected in 22%-36% among chronic liver disease groups. Strikingly, almost 83% of COMP-positive patients were cirrhotic at baseline, independently of cause of liver disease. Among the patients who developed HCC during follow-up, 73.7%(14/19) were COMP positive at baseline. COMP positivity was significantly associated with older age(P < 0.001), advanced fibrosis(P = 0.001) and necroinflammatory activity(P = 0.001), higher aspartate aminotransferase(P < 0.001), alanine aminotransferase(P < 0.02), γ-glutamyl transpeptidase(P = 0.003), alkaline phosphatase(P = 0.001), bilirubin(P < 0.05), international normalized ratio(P = 0.002) and alpha-fetoprotein levels(P < 0.02), and lower albumin(P < 0.001), and platelet count(P = 0.008). COMP levels [median(IQR)] were significantly higher in cirrhotics compared to non-cirrhotics [13.8(7.9) U/L vs 9.8(4.6) U/L, respectively; P < 0.001]. On multivariate logistic regression analysis, COMP-positivity was independently associated only with cirrhosis(OR = 4.40, 95%CI: 1.33-14.69, P = 0.015). Kaplan-Meier analysis showed that COMP positivity was significantly associated with HCC development(P = 0.007) and higher incidence of liver-related death(P < 0.001). CONCLUSION: Elevated COMP levels are strongly associated with cirrhosis and HCC progression. Serum COMP is a new promising non-invasive biomarker for HCC risk assessment in surveillance programs.