蛇毒检测技术研究进展

毒蛇咬伤仅从临床表现判断蛇种 ,常会误判 ,导致患者不能得到及时而正确治疗 ,因此 ,快速、准确的检测方法对判断蛇咬伤的蛇种是很重要的 ,而目前蛇毒的检测方法有生物检测法、免疫扩散法、免疫电泳法、放射免疫测定法、凝集测定法、荧光免疫测定法、酶联免疫吸附测定法等。而酶联免疫吸附测定法在专一性、敏感性、快速性、方便性方面取得了很大的进步 ,可用于野外检测。放射免疫测定法则运用于抗蛇毒 X因子的单克隆抗体 MP2来检测病人体液中的鲁塞尔蛇毒 ,这种方法的灵敏度在尿液中为 4ng/ml,在小牛血清白蛋白 -磷酸盐缓冲液中为 2 0 ng/…     

免疫测定法检测重金属技术的进展

作为检测重金属的一种新型方法,免疫测定法有其特点和优势。免疫测定法的技术构成包括重金属单克隆抗体的获得、单克隆抗体灵敏性的提高和免疫检测,其中重金属单克隆抗体的获得是基础。我们对该方法进行了简要介绍,并就其所面临的一些问题和发展趋势进行了探讨。     

苏云金芽孢杆菌毒力检测方法综述

苏云金芽孢杆菌是目前世界上研究最多、产量最大、应用最广的生物杀虫剂,它具有专一性强、对人畜无害、防治效果好以及生物降解无残毒等优点。本综合叙述了苏云金芽孢杆菌的毒力检测方法,包括生物测定法、HPLC法、免疫分析法、质谱法和电泳法,其中生物测定法是国际标准方法,已被普遍采用;HPLC法、免疫分析法、质谱法和电泳法是检测伴孢晶体或外毒素的含量,属于间接毒力测试方法。     

中空纤维测定法在抗肿瘤药物研究中的应用

中空纤维测定法是NCI建立的体内抗肿瘤药物筛选方法。本文介绍了中空纤维测定法的基本实验方法和疗效评判标准,回顾了近年基于中空纤维测定法的抗肿瘤药物方面的研究成果,认为中空纤维测定法不仅适用于药物筛选,在快速研究抗肿瘤药物药效和机制方面也具有重要的应用价值。     

ELISA法检测疫苗中牛血清残留量的适用性研究

为了验证ELISA法对病毒性疫苗中残余牛血清蛋白检测的适用性,用牛血清蛋白ELISA测定法与牛血清白蛋白ELISA测定法、牛血清IgG-ELISA测定法对麻疹疫苗、风疹疫苗、腮腺炎疫苗洗涤前病毒培养液、洗涤后病毒收获液以及多种成品疫苗中的残余牛血清蛋白含量进行了测定。结果显示,麻疹疫苗、风疹疫苗、腮腺炎疫苗洗涤前病毒培养液中牛血清白蛋白含量依次为IgG含量的70.5倍、65倍、84.3倍,洗涤后病毒收获液中两者比值分别为4.2、1.8和8.1;牛血清白蛋白ELISA法和牛血清蛋白ELISA法均能检测出疫苗中牛血清白蛋白,但前者测不出牛血清IgG,而后者可准确检测牛血清IgG。牛血清蛋白ELISA测定法可同时检测牛血清白蛋白和牛血清IgG,更适用于疫苗残余牛血清蛋白含量的测定。     

我国禾谷类病毒病的病原问题 Ⅸ.应用酶联免疫测定法检测小麦丛矮病植株

酶联免疫测定法(简称ELISA)是一种具有高度特异性和敏感性的检测方法。它的基本原理是:利用双功能试剂制备抗体(或抗原)与酶的结合物,该结合物保留了免疫学和酶的活性,酶与底物溶液产生的颜色反应可以定量测定。此法能在极低浓度下定量检测特异的抗原(或抗体),适用于疾病的大规模普查和流行病的调查。自E.Engvall 和Van Weemen 在1971年分别建立酶联免疫测定法以来,目前已广泛地应用于各种传     

镧系元素标记核酸探针技术

镧系元素标记核酸探针技术是利用某些镧系元素及其螯合物作为标记物,通过多种标记方法合成镧系元素核酸探针,用时间分辨荧光测定法进行检测,可以代替放射性核素标记探针进行各种检测和分析。该方法具有灵敏、快速、安全、简便、经济等特点。     

染色体辐射效应的微机检测 Ⅱ断片率 微核率与细胞畸变率间的相关性检测

本文继前文后,按照设计的线性回归程序在“IBM—PC/XT”微型计算机上,进一步检测了断片率、微核率与细胞畸变率之间的相关性,肯定了微核测定法,断片测定法可以替代染色体畸变分析法。     

SIV抗体简便快速免疫测定法

SIV(simian immunodeficiency virus)抗体测定法已在很多研究课题中被证明是有用的。但是在野外条件或设备条件差的实验室里很难开展常规免疫测定法。现已发展了一种价廉、简便、快速的免疫测定法,即用灭活SIV抗原和F-C试剂盒(美国加州E.Y药厂出品)检测SIV抗体。F-C试剂盒是一种薄膜滤纸装置,利用金标记A-蛋白测定抗体或抗原。这种低费用     

组织细胞一氧化氮含量测定的几种方法

一氧化氮(nitric oxide,NO)是一种新型的细胞信使分子,它在调节心血管系统、神经系统和免疫功能方面起着重要的作用.测定组织细胞 NO 的含量对于探讨NO 的生理功能具有重要的意义.该文简要介绍了应用化学发光法、微盘测定法、放射强度测定法和分光光度法检测组织细胞 NO 的含量.     

A fluorescence-based assay for indoleamine 2,3-dioxygenase

A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.     

Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor

Two assay methods for recombinant human tumor necrosis factor (rH-TNF) were developed, one a biological L-cell assay and the other an enzyme-linked immunosorbent assay. The accuracy and reproducibility of each and the correlation between the two were studied. As a result of this investigation, the two assay methods were found appropriate for standardization of rH-TNF. A freeze-dried reference was prepared, and examination of its potency and stability showed it to be suitable for use as a reference standard for rH-TNF assays.     

果胶酶分离纯化及分析方法的研究进展

果胶酶能降解果胶质,在果汁制造、果酒酿造等方面有着广泛应用。果胶酶分子生物学的迅速发展极大地促进了分离纯化与分析方法的研究。由于不同菌种产生的果胶酶成分复杂程度不同,分离纯化手段和分析方法也不相同。本文对果胶酶分离纯化手段及其分析方法进行了综述。     

Comparative evaluation of whole blood D-dimer test to plasma D-dimer test for diagnosis of disseminated intravascular coagulation

Three rapid D-dimer test methods were compared for the diagnosis of acute disseminated intravascular coagulation (DIC). These were (a) SimpliRED, an autologous red cell agglutination assay. (b) DIMERTEST latex agglutination assay, containing monoclonal antibody DD-3B6/22(6), and (c) D-DI latex agglutination assay containing mouse anti-human D-dimer monoclonal antibodies. The D-DI latex method having higher sensitivity (100%) and specificity (81%) in clinically acute DIC was postulated as the gold standard and compared with the other two methods. The results suggest that D-DI latex agglutination assay containing mouse anti-human D-Dimer monoclonal antibodies are the better assay methods amongst all the three kits analyzed. It is advisable to look for the nature of the antibody used to coat the latex particles in plasma based kits. In emergency setting RBC kits may be of some use as rapid diagnosis is advantageous.     

Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Evaluation of Assay Methods for Hepatitis-Associated Antigen

Assay methods for hepatitis-associated antigen (HAA) were evaluated for sensitivity, or reproducibility, or both in a series of three trials in which both research and service-oriented laboratories participated. Agar-gel diffusion (AGD) methods were found to be the least sensitive and reproducible of the commonly employed assay methods. Complement fixation (CF) tests were consistently more sensitive than either AGD or counterelectrophoresis (CEP) methods for detection of HAA. With judicious choice of the antibody reagent, sensitivity of CEP techniques was equivalent to CF methods of HAA detection. None of the three major assay methods (AGD, CEP, or CF) compared in this study were capable of consistently detecting HAA when it was present in relatively low concentrations in human serum.     

USE OF PCR ANALYSIS FOR STERILITY TESTING IN PHARMACEUTICAL ENVIRONMENTS

To determine the sterility of pharmaceutical samples, highly conserved bacterial ribosomal DNA sequences were used in a PCR-based assay. Finished products, raw materials, growth media, and diluents were artificially contaminated with different types of microorganisms. Samples were incubated for 24 h. After incubation, microbial DNA was extracted from enrichment broths using a Tris-EDTA-Tween 20 buffer containing proteinase K. Extracted DNA was added to Ready-To-Go PCR beads and eubacterial primers. Contaminated samples were found to contain the conserved 1.5 kilobase (kb) DNA fragment of the bacterial genome by using the PCR assay. None of the uninoculated samples was found to show the presence of the 1.5 kb fragment. PCR test results were compared with standard conventional methods. There was a 100% correlation between standard conventional methods and the PCR assay. However, the PCR-based assay was completed within 27 h while conventional methods required 4–5 days. Rapid PCR analysis using a simple sample preparation reduced the time for sterility testing of pharmaceutical samples allowing optimization of risk assessment and implementation of corrective actions.     

表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。