双抗体夹心ELISA法检测相思子毒素

目的：建立相思子毒素（abrin）的酶联免疫检测方法, 为abrin临床诊断、中毒治疗、法医学鉴定等应用领域提供技术基础和参考依据.方法：采用双抗体夹心ELISA法来检测微量abrin.结果：该法检测abrin线性范围为0.25 ～125 μg/L, 线性回归方程为Y=0.51015X＋0.4153（r=0.9880, n=10,P＜0.0001）, 检测下限为0.25 μg/L.不同浓度蓖麻毒素（ricin）、葡萄球菌肠毒素B（SEB）对检测结果基本无干扰.该法能用于abrin毒素污染水样、土样、食品、血液等模拟样品的分析, 相对标准差为3.52% ～4.86%, 具有较好的重现性.结论：成功地建立了双抗体夹心ELISA法检测abrin, 将多克隆抗体的强富集能力和单克隆抗体（mAb）的特异性结合起来, 提高检测的灵敏度和特异性, 可适用于各种微量abrin样品的分析.     

检测抗丙型肝炎病毒总抗体的双抗原夹心法的建立

以丙型肝炎病毒（ＨＣＶ）结构区和非结构区基因工程表达抗原及人工合成多肽包被酶标板，用辣根过氧化物酶标记抗原，建立了检测血清中抗－ＨＣＶ总抗体的双抗原夹心法。与普遍采用的检测抗－ＨＣＶＩｇＧ的间接酶联免疫吸附试验（ＥＬＩＳＡ）比较，其灵敏度（１∶１２８）稍高于间接法（１∶６４），特异性相同，均为１００％。该法可同时检测抗－ＨＣＶ各类抗体，使检出率提高１０％。     

双抗体夹心ELISA定量检测重组人干扰素α1b方法的建立与评价

目的 建立双抗体夹心ELISA法定量检测重组人干扰素α1b的方法.方法 筛选具有不同抗原结合位点的抗重组人干扰素α1b单克隆抗体,分别作为包被抗体和辣根过氧化酶标记抗体,建立双抗体夹心ELISA法定量检测不同批次重组人干扰素α1b含量,评价该法的检出限、精确度、重复性、特异性.结果 所建立的ELISA最低检出限为10 ng/ml,检测线性范围10～100 ng/ml,R2 =0.992,测定值与实际值偏差＞5％,板间变异系数均小于10％.结论 该方法灵敏度高,特异性强,准确性和重复性好,可用于重组人干扰素α1b成品的定量检测.     

建立基于核蛋白双抗体夹心ELISA的发热伴血小板减少综合征病毒滴定方法

目的 建立基于核蛋白双抗体夹心ELISA滴定发热伴血小板减少综合征病毒(SFTSV)的方法.方法 首先利用SFTSV核蛋白特异性多克隆和单克隆抗体建立双抗体夹心ELISA方法,用于SFTSV病毒滴度的检测,优化检测程序,评价检测方法的特异性与灵敏性,并与免疫荧光法和空斑试验法检测SFTSV滴度的方法进行比较.结果 所建立的基于双抗体夹心ELISA法滴定病毒与免疫荧光法和空斑试验法的滴定结果一致,相关系数分别为0.999和0.949.结论 基于SFTSV核蛋白双抗体夹心ELISA的滴定方法具有较高敏感性和特异性,操作简单,避免直接免疫荧光和空斑法滴定结果判定的主观性,并可适用于高通量检测.     

应用双抗体夹心ELISA检测人血清中Clq含量

本文采用抗人Clq单克隆抗体和多克隆抗体建立双抗体夹心ELISA,检测人血清中Clq含量。244份正常人血清标本检查结果,Clq正常值为287±56μg/ml。检测28份SLE患者血清有4份Clq呈明显下降.26份SLE病人血清同时用CH50试验检测总补体含量,发现二者呈正相关。因此,该技术有可能作为有关疾病如SLE等的临床诊断及疗效监测的辅助方法。     

朊蛋白N端和C端多肽特异性抗体的制备及ELISA检测技术的研究

目的 制备朊蛋白N端和C端多肽特异性抗体，并对ELISA方法在朊病毒病检测中的应用进行研究。方法构建人朊蛋白N端和C端多肽原核表达重组质粒，分别表达纯化融合蛋白。以此为抗原制备朊蛋白N端和C端多肽特异性抗体。ELISA和Western Blot检测所制备抗体与重组和天然的PrP蛋白的免疫反应性。初步建立间接ELISA检测技术。结果 所制备的N端和C端抗体可特异性识别重组全长PrP蛋白和相应的PrP片段，无明显交叉反应。C端抗体还可有效地识别感染羊瘙痒因子263K的仓鼠脑组织中经PK消化后的prp^Se，其Western Blot反应带型与PrP单抗3174相似。5000r／min离心处理脑组织悬液可有效保留上清中PrP^Se成分而不影响ELISA检测。蛋白酶K虽经灭活处理，但可明显抑制重组和天然PrP在液相中与相应抗体的结合。间接ELISA方法可根据反应A值区分正常或感染动物样本。结论 所制备的朊蛋白N端和C端抗体具有良好的特异性，C端抗体可用于实验性朊病毒病的检测。建立的间接ELISA方法可试用于朊病毒病的初步筛查。     

以ABTS为底物的双抗体夹心间接ELISA法的建立及应用

以多-单克隆抗体与正常鼠腹水平行包被固相载体,并用一种新的底物——ABTS作指示系统,建立了EHFV的双抗体间接夹心ELISA法。结果表明:1)ABTS与酶的反应平稳,不需加终止剂终止酶的反应,结果易于肉眼观察;2)对EHFV的定量测定较IFA客观、准确;3)对EHFV抗原的检出范围为351.65±122.82～2091.87±31.27ng/ml;4)对EHFV感染组织和细胞内EHFV的检出率为100％。以上结果说明该法敏感、特异、快速、简便、实用。并指出ABTS作为辣根过氧化物酶的底物的换代产品,应用前景广泛。     

双抗原夹心法ELISA在检测抗肾综合征出血热总抗体中的应用

目的建立可以检测不同来源血清中抗汉坦病毒抗体的简单、灵敏的方法。方法汉坦病毒核蛋白重组表达纯化后，同时作为捕获作用的固相抗原和检测作用酶标记抗原，建立检测血清中抗汉坦病毒总抗体的双抗原夹心法ELISA法，并与常用的IFA法进行比较分析。结果检测不同血清时特异性为100％，敏感性高于IFA法4～8倍。且不需考虑更换检测试剂，不同来源的血清样本对检测结果没有明显的影响。结论本方法操作简单，成本低，具有较高的敏感性和特异性，适合用于汉坦病毒感染的监测、调查和临床诊断以及宿主动物间病毒感染流行的调查与监测。     

TTV酶联免疫方法的建立及其初步应用

目的 建立检测TTV的酶免疫技术,了解不同型肝炎患者血清中抗－TTV抗体的分布情况,并结合TTV DNA的检测分析两者的关系。方法 以原核表达的TTV ORF1蛋白为抗原建立检测抗－TTV的酶联免疫吸附试验（ELISA）方法,采用该方法检测不同肝炎患者中抗－TTV抗体;在套式聚合酶链反应（PCR）方法检测血清标本中TTV DNA。结果 所建立的检测TTV的ELISA方法具有较好的特异性,不同别肝炎患者中抗－TTV抗体阳性率分别为：甲型肝炎患者10.5%（4／38）,乙型肝炎患者12.5%（16／128）,丙型肝炎患者8.3%(7/84),丁型肝炎患者7.7%(30/93),健康人群1.3%(1/78)。统计学分析表明,非甲－庚型肝炎患者抗－TTV阳性率显著高于其他型肝炎患者（P＜0.01）,而正常人群则显著低于肝炎患者（P＜0.05）。抗－TTV阳性率与TTV DNA阳性率存在相关性（P＜0.05）。结论 在不同型肝炎患者中均可检出抗-TTV抗体,但以非甲－庚型肝炎患者阳性率高;TTV抗体可与基因同时存在于患者血清中,抗－TTV抗体可能类似抗－HCV, 是一传染性标志。     

双抗体夹心生物素-亲和素ELISA法检测相思子毒素

目的 建立相思子毒素(abrin)的酶联免疫检测方法,为abrin临床诊断、中毒治疗、法医学鉴定等应用领域提供技术基础和参考依据.方法采用双抗体夹心生物素-亲和素ELISA法来检测微量abrin.结果该法检测abrin线性范围为0.125～31.25 μg/L,线性回归方程为y=0.52369X 0.51632(r=0.9816,P＜0.0001,n=9),检测限为0.125 μg/L.不同浓度蓖麻毒素(ricin)、葡萄球菌肠毒素(SEB)对检测结果基本无干扰,表明该法检测abrin具有很好的特异性.该法能用于abrin毒素污染水样、土样、食品、血液等模拟样品的分析,相对标准差为2.35%～4.14%,具有较好的重现性.结论 成功建立了夹心BA-ELISA法检测abrin,巧妙地将多克隆抗体的强富集能力、单克隆抗体的特异性以及生物素-亲和素系统的放大作用结合起来,达到了提高检测的灵敏度和特异性的目的 ,可适用于各种微量abrin样品的分析.     

抗人甲胎蛋白单克隆抗体的制备及双抗体夹心ELISA检测技术的建立

目的制备、筛选多株具有商用价值的可配对的高特异性、高亲和力的抗人甲胎蛋白(hAFP)单克隆抗体,初步建立双抗体夹心ELISA检测方法。方法通过经典的淋巴细胞杂交瘤技术筛选分泌抗hAFP单抗的杂交瘤细胞株;对筛选所得单抗的特性进行鉴定分析;双抗体夹心ELISA法筛选最佳配对抗体;初步建立DAS-ELISA检测方法并检测血样,绘制其标准检测曲线并与进口试剂盒比较。结果共获得12株稳定分泌抗hAFP单抗的杂交瘤细胞株,其中Ab 1~Ab 5 5株单抗的腹水效价均高于600万,Ab 5的效价高达3000万;特异性鉴定结果表明Ab 1~Ab 5均能够特异识别天然hAFP分子,但Ab 5与人血清白蛋白(HSA)存在较强交叉反应;抗体配对实验共筛选出5对(Ab 1/HRP-Ab 2、Ab 1/HRP-Ab 4、Ab 3/HRP-Ab 2、Ab 3/HRP-Ab 4和Ab 4/HRP-Ab 1)能够满足hAFP检测要求且无交叉反应的配对抗体,最终确定Ab 1+Ab 3/HRP-Ab 2和Ab 1+Ab 3/HRP-Ab 4为最佳配对组合;利用最佳配对抗体组合建立标准检测曲线,其线性检测范围为5~250 ng/ml,最低检测限2 ng/ml,检测上限400 ng/ml,优于进口试剂盒的线性范围5~200 ng/ml和最低检测限5 ng/ml;样检结果显示自制试剂盒的阳性血清检测准确率99.2%(119/120例),阴性血清准确率100%(40/40例)。结论筛选获得的4株高亲和力、高特异性抗hAFP单抗均可应用于DAS-ELISA试剂盒的研制,Ab 1和Ab 3适合作为捕获抗体,Ab 2和Ab 4适合作检测抗体;自制试剂盒的线性检测范围和最低检测限均优于进口试剂盒,表明具有商用价值。     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

双抗原夹心法检测抗HIV-1/2总抗体方法的建立和评价

目的 进一步提高HIv感染诊断试剂的敏感性。方法 以人工合成的HIv—1 gp41．1(sP1)、gp41．2(sP2)、gp120(sP3)、P24(sP4)和HIV—2gp36(sP5)5条多肽，采用双抗原夹心酶链免疫吸附试验(ELISA)原理，以sP1、sP3、sP4和sP5混合包被酶标板作为因相抗原，辣根过氧化物酶标记sp1、sp2、sP4和sP5多肽为标记物，建立了检测抗HIV—1／2总抗体的双抗原夹心ELISA法。结果 检测卫生部药品生物制品检定所第2代40份质控参比血清，其特异性和灵敏度均为100％，高于间接ELISA法(特异性为90％，灵敏度为65％)。检测210份其他病种患者血清均为阴性，与雅培HIVAB试剂比较检测如份健康献血员血清和88份HIv感染者血清，符合率为100％。结论 本方法特异性强、敏感性高，操作简便，适用于献血员的筛选和临床HIv感染的检测。     

The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5′ coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3′ UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5′ coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.     

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr≈30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8–3.3 and 3.0–3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose–response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r² of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319±237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.     

报告基因研究及其应用进展

报告基因是现代分子生物学研究领域中用于分析结构基因旁侧区域潜在的顺式元件(如启动子、增强子和沉默子等)和反式作用因子相互作用关系的一种重要工具,通过它的表达产物便捷、可靠、灵敏地解析基因时空表达调控的内在规律.报告基因技术广泛应用于基因表达调控、信号转导、转基因、启动子分析、受体功能鉴定、基因治疗以及药物筛选等领域,该文将对报告基因的主要类型、特点及其应用研究进展进行综述.     

ELISA法测定白细胞介素－6

建立了灵敏的双抗体夹心ＥＬＩＳＡ法测定人白细胞介素－６．主要步骤为以鼠抗重组白细胞介素－６单抗包被，加入待检抗原，再加多克隆兔抗重组白细胞介素－６血清作用，最后加酶标羊抗兔示踪并放大其反应。本法简便，快速，重复性好，灵敏度达１ｎｇ／ｍｌ，适用于临床与基础研究。     

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.