人精浆对抗原抗体反应免疫抑制作用的研究

本文采用体外实验的方法,研究人精浆对抗原抗体特异性反应的抑制作用。运用酶联免疫吸附试验原理检测人精浆对AsAb、A-dsDNA、A-TG、A-TM、RF五种自身抗体反应的抑制,抑制率分别为61.5±23.1(％)、57.1±19.4(％)、56.7±25.8(％)、42.0±28.2(％)、41.8±25.1(％),说明均有明显的抑制作用。经生育组与不孕组、流产组对比分析,发现不孕组精浆对RF反应的抑制率明显高于生育组,其它各组之间对比均无显著差异。本文还对精浆的免疫抑制作用进行了探讨。     

体外重组人细胞色素P450 2C9 酿酒酵母表达体系的构建及其基因多态性对药物相互作用影响的初探

 目的 体外构建重组人细胞色素 P450 2C9（CYP2C9）酿酒酵母（Saccharomyces cerevisiae）表达体系,且利用该体系研究药物对 CYP2C9 基因多态性酶抑制程度的差异性。 方法 将 CYP2C9*1（野生型）和通过定点突变 PCR 法获得的等位基因突变克隆 CYP2C9*2（R144C 突变体）和 CYP2C9*3（I359L 突变体）电穿孔转化酿酒酵母后,差速离心法制备酿酒酵母微粒体,蛋白免疫印迹法检测 3 种 CYP2C9 微粒体蛋白的表达;HPLC 法检测 CYP2C9*1 与特异性底物双氯芬酸的反应,记录产物峰面积值,利用 PrismDemo 软件计算米氏常数（Km）值;利用荧光底物 BOMCC 对 3 个等位基因进行酶活性分析,分别计算 Km、最大酶促反应速度（Vmax）和内在清除率。采用荧光高通量方法测定 5 种药物（甲苯磺丁脲、磺胺苯比唑、酮康唑、氟西汀和泰洛平）对酶的抑制程度。 结果 蛋白免疫印迹结果表明,3 个等位基因均表达目的蛋白。CYP2C9*1 代谢双氯芬酸的 Km 值为 5.34 ± 0.96 μmol/L;以 BOMCC 为底物时,CYP2C9*1 和 CYP2C9*2 的 Km 值分别为 16.94 ± 4.78、34.73 ± 5.51 μmol/L,Vmax 分别为 0.21 ± 0.10、0.12 ± 0.01 pmol/（min&;#8226;pmol P450）,内在清除率分别为 0.012 ± 0.003、0.003 ± 0.0001 µl/（min&;#8226;pmol P450）;CYP2C9*3 无催化活性。5 种药物对 CYP2C9*1 的抑制程度：磺胺苯比唑 > 酮康唑 > 氟西汀 > 甲苯磺丁脲 > 泰洛平;对 CYP2C9*2 的抑制程度：磺胺苯比唑 > 甲苯磺丁脲 > 酮康唑 > 氟西汀 > 泰洛平。 结论 成功构建了重组人细胞色素 CYP2C9 及其多态性等位基因（CYP2C9*2、CYP2C9*3）酿酒酵母表达系统;初步建立了药物对 CYP2C9 基因多态性酶的抑制作用体外检测体系,为指导临床联合用药奠定了基础。     

宫颈粘液中乳酸脱氢酶同工酶在月经周期和早孕期的变化及意义

为研究宫颈粘液中乳酸脱氢酶(LDH)同工酶在月经周期及早孕期间的变化规律.分别对正常月经周期期间和早孕妇女宫颈粘液标本60个和40个,采用琼脂糖凝胶电泳法分离LDH,以乳酸为底物的酶显色反应后,570nm处对电泳区带进行扫描,计算出各组分的百分含量.结果M-LDH在整个月经周期中约17天出现高峰、而H-LDH在整个月经周期中约11天出现高峰.在月经周期中M-LDH及早孕35-71天M-LDH的平均值分别为57.57±6.58%和49.14±7.47%,两者差异有非常显著意义(P＜0.01);月经周期中H-LDH及早孕35-71天H-LDH的平均值分别为18.06±3.40%和21.65±4.63%,两者差异也有非常显著意义(P＜0.01).结论确定宫颈粘液中LDH同工酶在月经周期及早孕期间的变化规律性及其差异,将对受孕、胚胎植入、胚胎发育等方面具有一定意义.     

青海撒拉族人外周血淋巴细胞A——醋酸萘酯酶反应阳性率及其亚群的观察

应用α-醋酸萘脂酶反应标记法,对94例青海撒拉族健康人的外周血淋巴细胞阳性率进行了研究.结果:α-醋酸萘酯酶反应者为T淋巴细胞.阳性率为75.15±5.22%,T淋巴细胞的亚群:“点状颗粒型”淋巴细胞为61.75±7.29%:“弥散颗粒型”淋巴细胞为13.08±3.39%。同时发现α-醋酸萘酯酶反应阳性淋巴细胞随年龄增长而发生变化.     

东北虎幼体血清酶含量的测定与分析

目的 测定东北虎幼体血液中8种血清酶含量,为东北虎的生长发育、繁殖及疾病诊治等积累数据,并为虎类亚种间遗传和进化关系等的研究提供参考。 方法 用HITCH 7600-20型全自动生化分析仪对13只东北虎幼体进行8种血清酶含量的测定。 结果 东北虎幼体血清中8种酶含量分别为：谷草转氨酶（AST）(29.31±5.88)IU/L,乳酸脱氢酶（LDH）(267.08±76.40)IU/L,羟丁酸脱氢酶（α-HBDH） (149.38±54.07)IU/L,谷酰转肽酶（GGT）(2.92±1.94)IU/L,肌酸激酶（CK）(284.77±132.02)IU/L,淀粉酶（AMY）(2 149.85±357.03)IU/L,肌酸激酶同工酶（CK-MB）(390.62±145.70)IU/L,乳酸脱氢酶同工酶（LDI）(11.11±7.08)IU/L。 结论 东北虎幼体雌雄个体间所测8种血清酶含量无显著差异,与同科动物猎豹、金钱豹比较在AST、LDH等酶类存在差异。     

HBV核心与前S1融合蛋白疫苗对HBV转基因鼠的免疫治疗效果观察

目的　观察乙型肝炎 (乙肝 )病毒核心与前S1融合蛋白疫苗 (HBVCS1)在HBV转基因小鼠中的免疫治疗效果。方法　 6只HBV转基因小鼠分成 2组 ,在 0周和 3周时实验组小鼠腹腔内注射HBVCS1与免疫佐剂 (弗氏完全佐剂 弗氏不完全佐剂 ) ,对照组仅注射弗氏完全佐剂与磷酸盐缓冲液PBS。采用 3H TdR掺入法检测转基因鼠脾淋巴细胞特异性增殖反应 ,以酶联免疫吸附试验检测血清中的乙肝病毒表面抗原HBsAg,采用荧光定量PCR检测血清中的HBVDNA。结果　实验组 (1 99± 0 35 )转基因鼠脾细胞抗原特异性增殖反应明显高于对照组 (1 5 6± 0 15 ) ;实验组第 6周 (3 5 8±0 80 )与第 9周 (3 4 5± 0 70 )血清中HBsAg终点滴度与第 0周 (4 36± 1 0 4 )及第 3周 (4 81± 0 98)以及同一时间实验组与对照组之间HBsAg终点滴度均差异有显著意义 ;实验组小鼠第 6周 (4 0 2± 0 2 9)和第 9周 (3 5 8± 0 17)血清中HBVDNA含量明显低于免疫前 (5 5 5± 0 88) ,而对照组各批血清无此差别。结论　HBVCS1蛋白疫苗在HBV转基因小鼠中能诱导特异性免疫 ,并具有抑制HBVDNA复制和HBsAg表达的治疗效果     

一种较理想的用于ELISA的辣根过氧化酶底物

比较了辣根过氧化酶(HRP)的三种还原底物,邻苯二胺(O-PD)、2,2′-连氮-双〔3-乙基苯并噻唑啉-6-磺酸盐〕(ABTS)及3,3′,5,5′-四甲基联苯胺(TMB)的动力学,发现(1)不论以游离 HRP、HRP-IgG 或 HEP-Fab′为酶制剂,其 Km 均以 TMB 为最小;(2)TMB 所需的 H_2O_2最适浓度最低;(3)以 TMB 为底物时,酶活力受 pH 的影响最小;(4)酶活力测定的灵敏度最高;(5)蓝色的氧化产物可用于酶标定位,而加酸停止反应后的黄色产物又可用于酶标免疫测定的定量比色。再加上三种化合物中,仅 TMB 为非致癌性,故 TMB 是一个比较理想的 ELISA 的 HRP 底物。     

运动对大鼠额叶和纹状体一氧化氮合酶阳性细胞的影响

目的　研究运动对运动中枢神经型一氧化氮合酶阳性细胞的影响。方法　将大鼠分为对照组和运动组 ,运动组大鼠游泳 5d ,用免疫组织化学方法检测额叶、纹状体一氧化氮合酶阳性细胞的变化。结果　运动组额叶、纹状体一氧化氮合酶阳性细胞的染色比对照组深 ,运动组额叶阳性细胞数量平均每张切片为 (7.2± 1.9)个 ,比对照组 (2 .4± 0 .6 )个明显增加 (P <0 .0 5 ) ,运动组纹状体阳性细胞数量平均每张切片为 (35 .0± 7.2 )个 ,也比对照组 (8.2± 1.3)个增加显著 (P <0 .0 1)。结论　运动增加了运动中枢一氧化氮合酶阳性细胞的表达     

低氧性肺动脉高压大鼠肺组织硫化氢的变化

本文旨在探讨低氧对大鼠内源性硫化氢体系的变化。采用生化反应方法测定血浆中硫化氢的含量和肺组织中硫化氢合酶的活性 ,采用定量竞争性RT PCR的方法检测肺组织中胱硫醚 γ 裂解酶 (CSE)mRNA的含量。结果显示 ,与对照组相比 ,低氧组大鼠的血浆硫化氢含量明显减少 [(1 92 2± 2 2 1 ) μmol Lvs (30 1 6± 32 4 ) μmol L ,P <0 0 1 ) ],肺组织中硫化氢合酶的活性明显下降 [(0 1 2 7± 0 0 2 3)vs (0 2 78± 0 0 99)nmol mgwettissue·min ,P <0 0 1 ],CSEmR NA的含量明显减少 [(1 0 2± 0 1 5 )× 1 0 - 6 fmolvs (2 1 7± 0 2 2 )× 1 0 - 6 fmol,P <0 0 1 ]。以上研究表明 ,低氧对大鼠的内源性硫化氢体系有抑制作用     

胃癌病人自身免疫机能的临床研究

本文报道我科近年来应用经典的NK 酶释放法和间接免疫荧光技术对30例进展期胃癌病人检测其自然杀伤细胞(NK)活性和T 细胞亚群,结果提示胃癌病人术前NK 活性明显低下,仅有1例近于正常值为35.7%,平均为16.70±1.40%,与正常值34.92±0.39%(n=50)比较有显著差异(P<0.     

Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Using a Synthetic Convergent Peptide Library, or Mixotope, for Diagnosis of Primary EBV Infection

An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive.     

Characterization of the humoral immune response to Chlamydia outer membrane protein 2 in chlamydial infection

Detection of antibodies to an outer membrane protein 2 (OMP2) by enzyme-linked immunosorbent assay (ELISA) by using either the Chlamydia trachomatis- or the Chlamydia pneumoniae-specific protein was investigated. OMP2 is an immunodominant antigen giving rise to antibody responses in humans infected with different C. trachomatis serovars (A to C and D to K) or with C. pneumoniae, which could be detected by OMP2 ELISA. OMP2 ELISA is not species specific, but antibody titers were usually higher on the homologous protein. The sensitivity of this assay was high but varied according to the "gold standard" applied. Levels of antibody to C. pneumoniae OMP2 as detected by ELISA seem to return to background or near-background values within a shorter period of time compared to antibodies to C. pneumoniae detected by microimmunofluorescence (MIF), making it more likely that positive results in ELISA reflect recent infection. Thus, OMP2 ELISA has distinct advantages over MIF and commercially available ELISAs and might be a useful tool for the serodiagnosis of chlamydial infection.     

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

Abstract

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (?5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

用协同凝集试验和SPA—固相免疫电镜技术检测EHFV抗原

本文建立了检测流行性出血热病毒(EHFV)抗原的协同凝集试验(COA)和SPA—固相免疫电镜技术(SPA—SPIEM)。COA检测粗制抗原时,先以正常鼠脑和肺悬液吸收用于致敏的抗血清,并用Cowanl株葡萄球菌预先处理抗原,可排除非特异性凝集反应。SPA—SPIEM似较COA敏感。研究结果提示COA和SPA—SPIEM均可用于EHFV抗原的检测,有可能被用于EHF疫区宿主流行病学调查和疾病的快速诊断。     

Study of the cultured human endothelial cells infected by epidemic hemorrhagic fever virus]

Virus antigen could be detected in the cytoplasm of infected human endothelial cells (HEC) by immunofluorescent assay (IFA) 2 to 10 days after the inoculation of epidemic hemorrhagic fever virus (EHFV), but no apparent histologic changes could be found by phase contrast light microscopy, as well as no mature virus particles could be detected under the transmission electron microscope. Reinoculation of the freeze-melt supernatant of HEC 8 days after the inoculation of EHFV to EHFV susceptible Vero E-6 cells, viral antigen could be detected in most of these cells and mature EHFV particles or viral inclusion bodies could also be obtained in the cytoplasm under transmission electron microscope. The results show that HEC is a susceptible target cell to EHFV and infection by this virus may not give apparent cytopathogenic effect in HEC.     

Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay

The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01?ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3)?IU/mL (χ(2)?=?25.04, p?相似文献   

A novel antigen detection immunoassay for field diagnosis of hepatitis C virus infection

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (approximately 5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

A novel separation-free assay technique for serum antibodies using antibody bridging assay principle and two-photon excitation fluorometry

A new technique for separation-free detection of antigen-specific antibodies is presented. The new technique employs antibody bridging assay principle and the recently developed ArcDia TPX fluorescence detection technology. According to the assay scheme, antibody molecules from the sample bind with one arm to an antigen on polymer microspheres and with the other arm to a fluorescently labeled secondary antigen reagent. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique, an assay for anti-adenovirus antibodies was constructed. The function of the assay method was tested both with monoclonal anti-adenovirus antibody preparation (standard analyte), and with positive serum samples. Standard class-specific ELISA was used as a reference method. The new assay method provides comparable sensitivity and precision, and wider dynamic range for IgG antibodies than the ELISA method. The standard curve showed linear response (R(2)=0.999) with a dynamic range of three orders of magnitude, detection limit (mean+3S.D.) of 8 pM, and intra-assay signal precision of 5%. Applicability of the new method for clinical serodiagnostics is discussed.     

Studies on IgG subclass antibodies in adult asthma. 1. Serum antigen specific IgG subclass antibodies in asthmatics with late asthmatic response

It is clear that immediate asthmatic response is mediated by IgE-dependent mechanisms. However, late asthmatic response is induced by inhalation of antigens without antigen specific IgE antibodies in some asthmatics, especially in intractable asthma induced by Candida antigen. To elucidate the relationship between those bronchial responses and antibodies, antigen specific IgG subclass antibodies in sera from asthmatics were measured and compared with IgE antibody. The results were as follows. 1. Avidin-biotin ELISA (enzyme-linked immunosorbent assay) method was established for the measurement of specific IgG and IgG subclass antibodies to mite or Candida antigen. 2. Serum levels of antigen specific IgG and IgG1 antibodies in asthmatics with LAR provoked by mite or Candida antigen were significantly higher than those in asthmatics without LAR (p less than 0.01). 3. Serum levels of specific IgE antibody to these antigens in asthmatics with LAR provoked by mite or Candida antigen were slightly lower than those in asthmatics without LAR, though the difference is not significant. These results suggest that high serum levels of specific IgG and IgG1 antibodies to these antigens play a role in inducing LAR in asthmatics with LAR.     

Hydroxyapatite-coated nylon beads as a new reagent to develop a particle agglutination assay system for detecting Japanese encephalitis virus-specific human antibodies.

BACKGROUND: Detection of Japanese encephalitis virus (JEV)-specific antibodies is done today by hemagglutination-inhibition assay (HIA), neutralization assay (NTA) and enzyme-linked immunosorbent assay (ELISA). These conventional assays are often difficult to perform in diagnostic laboratories with insufficient resources. An alternative antibody detection kit, which is simple, preservable and inexpensive, is needed for extended use in rural areas of Asia. OBJECTIVES: (i) Characterization of a new antigen carrier, hydroxyapatite-coated nylon (Ha-Ny) beads, and (ii) evaluation of the JEV antigen-coated Ha-Ny beads as a reagent to detect anti-JEV antibodies in human serum samples. STUDY DESIGN: We examined the Ha-Ny beads for hydroxyapatite content, precipitation efficiency and protein adsorption ability. We then developed a particle agglutination assay system using the JEV antigen-coated Ha-Ny beads, and tried out the newly developed assay system with reference serum samples. RESULTS: The beads had the ability to adsorb 0.44 mg of lysozyme per gram. Sedimentation speed was 10.2 cm/30 min in phosphate buffered saline (PBS), pH 7.0. Binding of the JEV antigen on Ha-Ny beads was confirmed by scanning electron microscopy (SEM) and ELISA. Eighteen confirmed-human serum samples were tested by the newly developed particle agglutination assay system. The results were consistent with those from HIA, NTA and ELISA. CONCLUSION: The Ha-Ny beads can be applicable to the development of a new JEV antibody-detection kit, which does not require specific laboratory facilities.