吖啶酯偶联单克隆抗体建立化学发光法检测黄曲霉毒素B1

[目的] 建立快速检测黄曲霉毒素B₁（aflatoxin B₁,AFB₁）的方法。[方法] 将吖啶酯（acridinium ester,AE）与AFB₁单克隆抗体进行体外化学合成,分别合成了吖啶酯∶AFB₁单克隆抗体摩尔比为5∶1、10∶1、15∶1、20∶1、25∶1的吖啶酯标记AFB₁单克隆抗体（AFB₁-AE）,分析了不同摩尔比的检测效果,选择吖啶酯∶AFB₁单克隆抗体为25∶1的AFB₁-AE偶联物建立化学发光免疫分析法（chemiluminescence immunoassay,CLIA）。[结果] 获得对应的标准曲线回归方程为：y=-0.245Ln（x）+ 1.578 1,R²=0.985 7,IC₅₀=81.48 pg/mL;对样品进行加标回收实验,加标回收率88.4%~106.0%,变异系数0.92%~2.10%。[结论] 将吖啶酯与AFB₁单克隆抗体体外交联,可建立新的化学发光免疫分析方法。     

酶联免疫法(ELISA)检测饲料中黄曲霉毒素B1

本文介绍了利用ELISA法的黄曲霉毒素B1(AFB1)试剂盒测定饲料中黄曲霉毒素B1的含量。样品经处理后,用甲醇水溶液提取饲料中黄曲霉毒素,提取液经过过滤、稀释后,用酶联免疫法直接测定。加标回收测定,平均加标回收率89%～93%,用酶联免疫法进行定性、定量测定,其特异性强,分析速度快,污染小,简化提取和纯化步骤,结果易判读。     

酶联免疫法检测饲料中黄曲霉毒素B1

黄曲霉毒素B1是危险的致癌物,经常在玉米、花生粕、棉籽粕和菜籽粕等饲料原料中检测到。经试验证明,使用酶联免疫法检测黄曲霉毒素B1灵敏度高,最低检出限为1μg/kg;标准曲线r值≥0.9955,变异系数≤3.5%,检测回收率≥95.02%;此外还具有检测时间短,检测过程约需2h,重复性好,操作过程简单方便,不接触有毒试剂等优点。     

酶联免疫法检测黄曲霉毒素B1的关键点

1黄曲霉毒素简介 1.1黄曲霉毒素的产生原因 黄曲霉毒素主要是由可以产生黄曲霉毒素的霉菌产生，霉菌生长繁殖需要一定的温度、湿度条件。如产生黄曲霉毒素的温度范围为12℃～41℃，其中最适宜的温度为25℃～32℃。相对湿度为86％～87％条件下，在48h内黄曲霉能很快生长。     

采用酶联免疫吸附法测黄曲霉毒素B1影响因素分析

采用酶联免疫吸附法（ELISA）检测饲料、食品时，样品的含盐量、酸碱性、金属离子含量，以及提取液中甲醇含量都对测定结果有一定的影响，而用氯仿（三氯甲烷）作提取液则将消除这些影响。     

黄曲霉毒素B1免疫层析检测方法的研究

以柠檬酸三钠为还原剂,制备了20nm胶体金颗粒,以此胶体金标记纯化的单抗4G6,喷于玻璃纤维上,AFBl蛋白偶联物和羊抗鼠IgG分别结合于硝酸纤维膜上,依次将样本垫、胶金垫、硝酸纤维膜和吸水纸组装,切割成胶体金试纸条。结果：组装的试纸条特异性好,与AF类似物无交叉反应,对AFBl标准品最低检测限为3μg／L,检测时间约为10min,批内和批间重复性为100％,假阳性率和假阴性率均为0。操作简便,成本很低,适于AFBl的现场快速检测。在制备了单克隆抗体的基础上,应用胶体金免疫层析技术,建立了食品中黄曲霉毒素B1（Aflatoxin B1,AFBl）快速检测方法。     

酶联免疫法检测饲料中黄曲霉毒素B1

本文采用ELISA法测定黄曲霉毒素B1。本法检测线性范围0.1～10ng/ml,最底检出浓度0.1ng/ml,回收率87%～95%。本法具有快速、简便和一次可批量定性定量的优点,适用于饲料等样品的黄曲霉毒素B1的分析测定。     

酶联免疫法对生鲜乳中黄曲霉毒素M1的快速检测

采用酶联免疫法（ELISA）对生鲜乳中黄曲霉毒素M₁进行检测,对比了3个不同厂家的试剂盒检测结果。结果表明,在空白样品中添加浓度为0.5、1.0、2.5 μg/kg时,试剂盒的回收率在93.5%～103.5%。不同厂家的试剂盒测定结果重复性、精确度均较好,说明酶联免疫法能够有效检测生鲜乳中黄曲霉毒素M₁。     

锦阳市饲料黄曲霉毒素B1污染情况调查

本次调查利用固相酶联免疫吸附(ELISA)原理,对在四川省绵阳市生产、销售、使用的饲料产品进行抽样,取得155份样品进行黄曲霉毒素B1含量检测.结果表明,绵阳市饲料黄曲霉毒素B1检出率为100％,总体超标率为3.9％,但乳(仔)猪饲料黄曲霉毒素B1超标率为6.2％,相对其他类型饲料较高,方差分析显示配合饲料与浓缩饲料的黄曲霉毒素B1污染情况差异显著(P＜0.05).     

Development and evaluation of an indirect enzyme immunoassay for detection of porcine antibodies to pseudorabies virus.

An indirect enzyme immunoassay is described for detection of porcine serum antibody to pseudorabies virus. The analytical sensitivity of the enzyme immunoassay was found to be approximately 4.5 log 4 X 10 (5120 times) greater than the serum neutralization test, based on parallel end point titrations. The diagnostic sensitivity of the enzyme immunoassay was comparable or superior to that of the serum neutralization, based on the earliest detectable antibody after infection of swine with pseudorabies virus by intranasal or intrauterine routes or by contact with infected pigs. The enzyme immunoassay, at a screening dilution of 1:20, gave 100% agreement with ELISA results provided with a U.S. Department of Agriculture-Animal and Plant Health Inspection Service proficiency panel of 40 sera. One serum having demonstrable antibody by the enzyme immunoassay was seronegative by the serum neutralization test.     

间接竞争ELISA检测TGEV方法的建立

猪传染性胃肠炎(transmissible gastroenteri-tis,TGE)是由冠状病毒科猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)引起的以仔猪呕吐、腹泻、严重脱水为特征的消化道传染病。病毒粒子随粪便大量排出,是造成病毒扩大传播、仔猪大批死亡的重要原因。采取粪便样品进行病原检测,对该病早期诊断具有重要意义。目前针对TGEV感染有多种快速诊断方法,概括起来可分为针对病毒结构蛋白进行的各种免疫学诊断技术和近些年发展的以病毒核酸为基础的分子生物学检测方法。其中免疫学方法以其特异性强、易操作、不需要昂贵仪器等特点…     

伏马菌素FB2间接竞争ELISA检测方法的建立

用卵清白蛋白与伏马菌素B2的偶联物(OVA-FB2)做包被抗原,标准伏马菌素B2(FB2)做竞争抗原,初步建立了检测玉米粉中伏马菌素B2的间接竞争ELISA方法.优化后的ELISA检测方法,线性范围为7.81～250μg/L,最低检测限为6.09 μg/L.曲线回归方程为y=-47.038x+120.25,R2=0.983 5,批内平均变异系数为3.92%,批间平均变异系数为5.53%.对玉米粉加标样品测定结果表明,利用甲醇-EDTA法提取样品的平均加标回收率达到了96.11%.     

莱克多巴胺单克隆抗体间接竞争ELISA检测方法的建立

用混合酸酐法将莱克多巴胺(Rac)与匙孔蓝蛋白(KLH)、牛血清白蛋白(BSA)偶联,经紫外光谱扫描确定偶联成功,测得偶联物Rac-KLH浓度为3.6 mg/mL,Rac-BSA浓度为6.2 mg/mL。以偶联物Rac-KLH作为免疫原,免疫6～8周龄雌性BALB/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/O-Ag-14进行融合。以Rac-BSA作为包被抗原,经间接ELISA筛选出阳性细胞株,再用有限稀释法进行多次亚克隆,获得稳定分泌单克隆抗体的杂交瘤细胞株1D9、1F3、2C11、5C3、3A10、4A8、6B7、6D5、7C11、7D3。其中单克隆抗体5C3和3A10间接ELISA效价1∶500 000,对莱克多巴胺的半数阻断浓度(IC50)均为3.98 ng/mL,对盐酸克伦特罗和沙丁醇胺的IC50均大于2 000 ng/mL;对盐酸克伦特罗和沙丁醇胺的交叉反应率均小于0.2%。间接竞争ELISA检测莱克多巴胺在0.5～100 ng/mL范围内为线性分布,确定的最低检测限为0.5 ng/mL。     

Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA.     

氟喹诺酮类药物多残留间接竞争ELISA检测方法的建立

为建立同时检测多种氟喹诺酮药物的免疫学分析方法,本研究以碳二亚胺(EDC)二步法合成环丙沙星人工抗原,免疫新西兰大白兔制备多克隆抗体,建立氟喹诺酮类药物多残留检测方法.标准曲线在PBS中的线性检测范围为0.2 ng/mL～376 ng/mL,半数抑制浓度(IC50)为8 ng/mL,检测限(LOD)为0.1 ng/mL;抗体对恩诺沙星、诺氟沙星和培氟沙星均能特异性识别,IC50值分别为7.3 ng/mL、10.6 ng/mL、和13.2 ng/mL;标准品稀释液中NaOH和甲醇的最高容忍度分别为10％和30％;牛奶基质中添加5 ng/mL、20 ng/mL和50 ng/mL的标准品,环丙沙星、恩诺沙星、诺氟沙星和培氟沙星的回收率分别为93％～108％、96％～110％、92.5％～104％和93％～102％.     

氟喹诺酮类药物多组份残留的酶免疫分析法

采用N-羟基琥珀酰亚胺(NHS)活性酯法制备诺氟沙星、环丙沙星、达氟沙星和沙拉沙星完全抗原,经紫外光谱扫描鉴定后,免疫小鼠,ELISA测定抗体交叉反应性,筛选簇特异性抗体,建立多组份ciELISA检测方法.紫外光谱扫描结果证实4种半抗原与载体蛋白发生共价结合,所制备的沙拉沙星与诺氟沙星抗血清效价都在1:64 000以上,环丙沙星和达氟沙星抗血清效价都在1 :32 000以上.交叉反应率测定结果表明,沙拉沙星抗血清对沙拉沙星、诺氟沙星、环丙沙星和恩诺沙星的交叉反应率依次为100.00%、92.68%、89.12%和83.56%;利用沙拉沙星抗血清建立了针对上述4种药物的多组份ciELISA检测方法,最低检测限依次为19.3、34.5、31.3、29.2 μg/L,大于5μg/L水平的添加回收率均大于83%.     

家禽饲料中玉米赤霉烯酮化学发光酶联免疫分析方法的建立

为了建立比较间接竞争化学发光酶联免疫分析法(ic-CLEIA)与直接竞争化学发光酶联免疫分析法(dc-CLEIA)检测家禽饲料中玉米赤霉烯酮(zearalenone,ZEN)的方法,采用二因子交叉试验优化确定最佳包被原浓度和抗体稀释度,建立ic-CLEIA和dc-CLEIA。结果显示:icCLEIA在0.05~5 ng/m L范围内线性关系良好,线性回归方程为y=-0.383 5x+0.417 9(R2=0.994 6),灵敏度(IC50)为0.611 ng/m L,检出限(LOD)为15 pg/m L,平均批内和批间变异系数为1.79%和1.02%,家禽饲料添加样品回收率为91.9%~112.1%,变异系数小于10.5%;dcCLEIA在0.025~5 ng/m L范围内线性关系良好,线性回归方程为y=-0.386 5x+0.334 1(R2=0.991 8),灵敏度(IC50)为0.372 ng/m L,检出限(LOD)为3 pg/m L,平均批内和批间变异系数为1.16%和1.20%,添加样品回收率为87.3%~119.3%,变异系数小于9.8%。结果表明:两种方法精确,灵敏度高,均可作为定量检测ZEN的有效手段;与ic-CLEIA相比,dc-CLEIA更适用于检测家禽饲料中的ZEN。     

Application of the indirect enzyme immunoassay for the detection of antibodies against Erysipelothrix rhusiopathiae

Sera from swine and rats experimentally infected with Erysipelothrix rhusiopathiae and field sera from swine were investigated for antibodies against E. rhusiopathiae using the microtiter enzyme immunoassay (EIA) and, for comparison, the growth test (GT) and the agglutination test (AT). In principle there was a good correspondence between the results of EIA and those of the two other methods, but EIA and GT were more sensitive than AT. On the basis of the evaluation pattern of GT and AT on swine sera, EIA titers of 1/320 were considered as “chronic erysipelas titers”. Compared with GT and AT, the EIA has some advantages: it is not influenced by contamination of the test sera, it takes only a few hours and using the microtiter system it is easy and economical to perform.