检测人IL—3夹心ELISA方法的建立

人ＩＬ3是一种作用于早期造血干细胞,影响各系血细胞增殖和分化的造血细胞因子[1,2]。临床上某些造血系统疾病可能与ＩＬ3的表达异常有关[3]。目前,应用依赖细胞系所建立的ＩＬ3检测方法,不能较好地鉴定和区分ＩＬ3与ＧＭＣＳＦ[4]。而应用骨髓细胞软琼脂培养计数其克隆形...     

D2蛋白酶双抗体夹心ELISA定量检测方法的建立

目的:建立一种定量测定D2蛋白酶的双抗体夹心ELISA方法.方法:用棋盘滴定法确定捕获抗体、检测抗体的最佳工作浓度;绘制标准曲线,确定线性范围、检测限和定量限;检测该方法的准确性(回收率)、精密性和特异性,并与Bradford法检测结果进行比较.结果:包被抗体和捕获抗体的最佳包被效价分别为1:4 000和1:5 000;检测的线性范围为(28～1180)μg/L,检测限为4.6μg/L.经方法学考核,批内、批间变异系数分别为5.64%和7.17%,回收率为92.44%～105.26%,与碱性蛋白酶、蛋白酶K等其他蛋白无交叉反应,与Bradford法检测结果具有良好的相关性.结论:该方法灵敏度高,重复性好,为后期D2蛋白酶规模发酵优化、分离纯化研究提供了定量检测的方法,并为后期药代动力学、临床研究提供了思路和备选方法.     

检测IV型胶原蛋白双mAb夹心ELISA的建立

慢性肝病通常发展为肝纤维化 ,常规实验室检查对早期肝纤维化的诊断敏感性差 ,且特异性不强。因此 ,建立一种可定量分析肝纤维化的方法尤为重要。血清Ⅳ型胶原蛋白(ＣＯＬⅣ )的水平与肝纤维化的程度密切相关 ,并可作为肝纤维化早期诊断指标之一[1,2 ] 。因此 ,我们采用两株抗ＣＯＬⅣ的单克隆抗体 (ｍＡｂ) ,建立了一种特异性强、灵敏度高、操作简便 ,适于临床检测血清ＣＯＬⅣ水平的双ｍＡｂ夹心ＥＬＩＳＡ法1　材料和方法1.1　材料　血清样品 4 9份 ,30份来自经体检排除心、肝、肾疾病的志愿者 (男女各半 ,年龄 2 6～ 5 0岁 ) ;19份采自…     

人内皮唾液酸蛋白改良双抗体夹心ELISA检测方法的建立

为建立肿瘤内皮标志物1(tumor endothelial marker 1,TEM 1)ELISA检测方法,依据TEM 1基因胞外区片段序列及原核表达载体pMAL-p5x多克隆位点设计引物,采用PCR技术从含TEM 1全长基因载体中扩增目的片段,将双酶切的目的基因和载体经胶回收连接后插入表达载体并经测序鉴定,在转入宿主菌后用IPTG诱导目的蛋白表达,用分离纯化后的目的蛋白分别免疫鼠和兔以制备多克隆抗体并用ELISA和流式细胞术对抗体进行鉴定,采用兔抗TEM1为捕获抗体、鼠抗TEM 1为检测抗体建立双抗体夹心ELISA检测方法,棋盘滴定法探索各抗体最佳工作浓度,倍比稀释TEM 1,检测建立方法的灵敏度。成功构建的重组表达质粒pMAL-p5x-hCD248,经纯化复性后获得目的蛋白,免疫后获得兔多抗抗体效价为1∶1 024 000、鼠多抗抗体1∶512 000,棋盘法确定捕获抗体、检测抗体及酶标抗体最佳浓度分别为1∶500、1∶16 000和1∶20 000,经检测证实该方法测定下限为18.31ng/mL;检测50例恶性肿瘤患者与50例健康人血清TEM 1含量,发现差异无统计学意义。     

A型肉毒毒素双抗夹心ELISA检测方法的建立

目的：建立A型肉毒毒素双抗夹心ELISA检测方法。方法：以高亲和力人源抗体ML01作为捕获抗体,以小鼠腹水诱生法制备的鼠源抗体BAS46为检测抗体,HRP标记的羊抗鼠IgG为二抗,建立A型肉毒毒素双抗夹心ELISA检测方法,并评价其灵敏度、重复性、检测线性范围和加样回收率等。结果：A型肉毒毒素双抗夹心ELISA检测法的灵敏度为2.56 pg/ml,变异系数为3.183%～12.030%,线性范围为0.244～62.500 ng/ml,回收率为90%～110%。结论：成功建立了A型肉毒毒素双抗体夹心ELISA检测方法,该方法快速、有效。     

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。     

检测抗伤寒杆菌抗体的双抗原夹心ELISA法

<正>目前肥达氏试验结果仍作为伤寒的诊断依据,缺点是敏感性低和特异性差。国内学者对此曾作改进,但仍欠理想。我们应用超声粉碎法制备的伤寒杆菌可溶性抗原,建立了双抗原夹心ELISA法用于检测血清中抗伤寒杆菌抗体,现报告如下。     

测定尿激酶含量夹心法ELISA的建立

采用两种识别不同抗原决定簇的抗尿激酶(UK)单克隆抗体建立了测定人 UK 含量的夹心法ELISA.本法灵敏度为0.15ng/ml,批内 CV4.3%,批间 CV8.7%,平均回收率98%.应用本法测定了部分正常细胞和肿瘤细胞株培养上清中的 UK 含量,肿瘤细胞培养上清 UK 量明显高于正常细胞.测定82名正常人血浆中 UK 含量,其结果为1.31±0.6ng/ml.     

双抗体夹心ELISA法检测人血清弓形虫抗体

本文报告了双抗体夹心竞争ＥＬＩＳＡ法检测人血清弓形虫抗体，并与间接ＥＬＩＳＡ法进行了比较。结果二法阳性符合率为９１．７％。夹心ＥＬＩＳＡ法特异性、稳定性优于间接ＥＬＩＳＡ法，间接ＥＬＩＳＡ法阳性检出率略高于夹心法，但无统计学差异（Ｐ＞０．０５）。     

单克隆抗体ELISA双抗体夹心法测定人白细胞介素2

本文叙述了测定人白细胞介素2(IL-2)含量的ELISA双抗体夹心法。用辣根过氧化物酶标记IL-2单克隆抗体。聚苯乙烯反应板预先用0.1％戊二醛处理,然后以单克隆抗体包被。本法批内变异系数CV=3.99％,批间变异系数CV=5.96％,标准曲线的测定范围为3.1～100u/ml,检测下限为1.55u/ml。应用本法已测定了2S例正常人和11例恶性肿瘤患者的IL-2含量。     

一种检测可溶性白细胞介素2受体夹心法ELISA的建立

采用两种识别不同表位的小鼠抗人白细胞介素2受体(IL-2R)单克隆抗体(McAb),在国内首次建立了检测可溶性IL-2R(sIL-2R)的夹心法ELISA,并进行了初步应用。结果显示:本法特异性强、敏感性高、重复性好、操作简便,能检出正常人、某些患者血清中以及细胞培养上清中sIL-2R,可用于基础和临床免疫学研究。     

Quantitative double antibody sandwich ELISA for the determination of gliadin

A sensitive double antibody sandwich ELISA (DAS-ELISA) based on chicken anti-gliadin IgY and biotinylated monoclonal antibody (mAb) was developed for the quantification of gliadin in foods. The anti-gliadin IgY and mAb specifically detected gliadin in wheat, barley, and rye by indirect ELISA and Western-blot assay. Using anti-gliadin IgY as capture antibody and biotinylated mAb as detecting antibody, the sensitivity of DAS-ELISA has a linear standard range of 4-40?ng/mL, showing that the limit of detection (LOD) corresponds to 4?ng/mL gliadin in assay buffer, equivalent to 0.8?ppm in foods. The intra-assay expressed as percentage of coefficients of variation (%CV) was 7.25% average of six food samples. The interassay precision was 9.51% in food samples. The combination of anti-gliadin IgY and biotinylated mAb in the DAS-ELISA provides a reliable, sensitive, and inexpensive tool for the detection of gliadin in gluten-free and gluten-containing food products.     

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40?ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.     

检测禽流感病毒抗原的双抗体夹心ELISA方法的建立

禽流行性感冒（avian influenza,AI）简称禽流感,是由正粘病毒科流感病毒属A型流感病毒引起的禽类传染病。高致病性禽流感（highly pathogenic avian influenza,HPAI）被世界动物卫生组织（world organization for animal health,OIE）列为A类传染病。我国也把禽流感列为一类动物疫病。禽流感病毒（avian influenza virus,AIV）可感染几乎所有野生禽及家禽,     

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.     

Development of a double monoclonal antibody sandwich ELISA test for Verticillium dahliae Kleb.

Verticillium dahliae causes wilt diseases in a wide range of horticultural and field crops in many parts of Turkey. In the Aegean region it has been a serious problem in cotton and vegetables for many years. More recently, it has become a major problem for olive growers in relatively newly established orchards. The fact that the only possible control methods of Verticillium wilt disease is the use of healthy and pathogen free propagating material has directed us to produce monoclonal antibodies against V. dahliae in order to apply rapid, sensitive and reliable serological detection methods. Verticillium spp. isolates were obtained from olive plantations, as well as from cotton and tomato fields in the Aegean and Marmara Regions. All suspected isolates were obtained as V. dahliae after determining microscopic morphological characteristics and pathogenicity tests on cotton seedlings. Immunizing antigens were prepared by three different methods including surface washing system, czapek dox agar and gel filtration methods. BALB/c mice were immunized with each antigenic form. Lymph node, spleen and bone marrow cells were used as sources of B-lymphocytes and 8D2 (IgM) and 7D6 (IgG1) were obtained from the spleen and lymph node fusion. The monoclonal antibodies were purified and immunoglobulin types were identified. 8D2 monoclonal antibody gave positive reaction with the V.dahliae isolates from olive, cotton, tomato and watermelon; however, it didn't give any cross reactivity with other epiphytic fungi. 7D6 antibody displayed cross-reactions with a few fungi. The monoclonal antibody (8D2) was conjugated with horseradish peroxidase (HRP). These monoclonal antibodies were characterized for use in the development of diagnostic kits based on double-monoclonal antibody sandwich ELISA test system for detecting V. dahliae in Turkish isolates. In this test, the first antibody was used as capture antibody and the second one was used for detection of antigens.     

Comparison of indirect double antibody and double antibody sandwich ELISA techniques with latex agglutination test for the diagnosis of human rotavirus infection

94 faecal samples from infants and children suffering of acute gastroenteritis were investigated for rotavirus by indirect double antibody sandwich ELISA kit (WHO, Geneva), Rotavirus ELISA kit (DAKOPATTS A/S, Copenhagen) and Rotalex latex-agglutination kit (Orion Diagnostica, Helsinki). The ELISA techniques gave almost identical results and seemed to be of same sensitivity and specificity. Rotalex agglutination had an overall agreement of 88% with ELISA. It is concluded that strongly positive reactions found by Rotalex may be regarded as true positive reactions, whereas samples producing weakly positive and/or negative reactions should be retested in a more specific and sensitive assay, such as enzyme linked immunosorbent-assay (ELISA).     

氯霉素乙酰基转移酶双抗体夹心ELISA检测方法的建立

目的 建立氯霉素乙酰基转移酶（chloramphenicol acetyltransferase,CAT)双抗体夹心ELISA的实验室检测方法。方法 从质粒pBLCAT6经PCR扩增CAT基因序列，插入到原核表达质粒pGEX-2T中，在大肠埃希菌DH5α中诱导表达；以纯化的融合蛋白GST-CAT为抗原免疫实验用兔，得到的CAT抗血清进行生物素标记，并在此基础上利用生物素链和亲和素放大系统建立双抗体夹心ELISA法，构建不同YY1结合位点突变的HPV16LCRCAT报道质粒，体外瞬时转染真核细胞，提取胞质蛋白，以建立的方法进行CAT表达检测。结果 SDS-PAGE显示表达的GST-CAT融合蛋白相对分子质量约为54000；制备的CAT抗血清可有效地识别原核及哺乳动物细胞表达的CAT蛋白；在不同启动子及调节序列的控制下，利用建立的CAT检测技术证明在瞬时转染的细胞中可诱导2-8倍的CAT表达增强。结论 建立的双抗体夹心ELISA方法可敏感地反映上游序列的启动子活性，以及启动子调节序列变化对启动子活性的影响，使CAT作为报道基因的研究更为简单化。