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。     

蛋白酶K单抗研制及其双抗体夹心ELISA定量检测方法的建立

目的 研制蛋白酶K单克隆抗体并构建定量检测蛋白酶K的双抗体夹心ELISA方法 ,用于生物制品中蛋白酶K的检测。方法 用杂交瘤技术制备抗蛋白酶K的单抗,用棋盘法优化ELISA条件,建立定量检测蛋白酶K的双抗体夹心ELISA方法,分析其特异性、精密性和准确性。结果 经免疫、融合、克隆、筛选到稳定分泌蛋白酶K单抗的杂交瘤细胞。获得1株阻断蛋白酶K活性的单抗5G6,以5G6为包被抗体、4D3为酶标抗体,构建蛋白酶K定量检测的ELISA方法。本方法线性相关系数R=0.99,线性范围为293.4~2 500 pg/ml;定量限度为293.4 pg/ml;变异系数为CV<15%;回收率介于89.3%~129.2%之间,37℃6 d的回收率>80%;与蛋白酶K以外的其他蛋白无交叉反应。结论 获得阻断蛋白酶K活性的单抗,构建出灵敏、特异的蛋白酶K定量检测方法。     

新型冠状病毒抗原检测双抗体夹心ELISA方法的建立

目的:探索即时、快速、准确的新型冠状病毒(2019-nCoV)抗原检测方法,提高新型冠状病毒感染者检出率,并建立2019-nCoV的S蛋白特异性检测方法。方法:本研究利用ELISA方法对前期研发的2019-nCoV基因工程单克隆抗体(mAb)进行配对检测,从中挑选出最优检测抗体组合,建立了新型冠状病毒抗原检测双抗体夹心...     

测定人血清纤维连接蛋白含量的双抗体夹心ELISA法的建立

４株经细胞融合获得的针对纤维连接蛋白（Ｆｎ）的单克隆抗体，经ＥＬＩＳＡ抗体相加试验和抗体竞争试验证实，为作用于Ｆｎ不同位点的单克隆抗体。选择其中２株单克隆抗体，应用于测定人血清Ｆｎ含量的双抗体夹心ＥＬＩＳＡ法．其批内差异为４．５％，批间差异为５．９％．建立的标准曲线相关系数为０．９８２，测定１３２例献血员血清Ｆｎ合量为０．２５５±０．０２８ｍｇ／ｍｌ，与文献报道相符．     

白念珠菌感染特异性Csa2蛋白双抗体夹心ELISA体系的建立与评价

目的以白念珠菌Csa2蛋白为靶标,建立双抗体夹心ELISA检测体系,评价该方法的检测灵敏度和特异性。方法构建pPIC9K-Csa2重组表达载体,电转化毕赤酵母GS115,甲醇诱导表达、纯化重组蛋白rCsa2。rCsa2蛋白分别免疫新西兰大白兔和豚鼠制备免疫血清。以纯化兔多抗和豚鼠抗血清配对,利用棋盘滴定法,确定最佳实验条件,建立双抗体夹心ELISA检测系统。检测rCsa2和白念珠菌不同培养时间的上清,确定检测方法的灵敏度;检测其他3种念珠菌、5种曲霉、新型隐球菌和马尔尼菲青霉的培养上清,评价检测方法的特异性。结果成功构建表达载体并获得真核表达重组蛋白rCsa2,经SDS-PAGE鉴定相对分子质量(Mr)为13 300,符合预期值;Western blot法证实该蛋白可与特异性抗体结合。免疫动物获得高效价免疫血清,并以此成功建立双抗体夹心ELISA,可检测rCsa2蛋白的灵敏度约为240 pg/mL,并最早可检测到培养18 h的白念珠菌培养上清,与其他10种临床常见真菌的培养上清无交叉反应。结论建立了有较高的检测灵敏度和特异性的Csa2的双抗体夹心ELISA,为区别诊断白念珠菌感染提供新的方法。     

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）。     

汉城病毒L99株双抗体夹心ELISA抗原检测方法的建立及应用

目的:建立汉城病毒(Seoul virus, SEOV)L99株特异性双抗体夹心ELISA抗原检测方法,验证其检测性能,为双价肾综合征出血热(hemorrhagic fever with renal syndrome, HFRS)疫苗研制、生产和检定过程中Ⅱ型病毒抗原含量检测提供有效手段。方法:小鼠体内诱生法制备L99...     

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HBsAg单克隆抗体在诊断中应用错位点双单克隆抗体ELISA夹心法的建立

对5种过氧化物酶标记的抗HBsAg单克隆抗体和14种固相物包被用单克隆抗体做了筛选试验，以探讨实际应用中各单克隆抗体的较佳配用方案。试验结果表明，用正交法所得的安验结果中，最佳配用情况下，包被用抗体和主要的酶标记抗体的作同位点是错位的。即捕捉抗体与标记抗体分别作用在HBsAg的不同位点上，两者之间基本上没有位阻现象。这种双单克隆抗体央心去的本底很低．可以目测判定结果。敏感性和特异性较好。HBsAg阳性，滴度为1：16(对流电泳法)的患者血清稀释至2^19时仍可得到P／N比≥2．1的读数，目测判定结果对至少在2^18稀释度下可明确地判定出阳性结果。     

Highly potent,naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

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重组人源细胞珠蛋白单抗制备及检测方法建立

目的制备重组人源细胞珠蛋白（recombinanthumanCytoglobin,rhCygb）单克隆抗体,并建立检测该蛋白双抗体夹心ELISA法,为下一步研究rhCygb药代动力学做准备。方法用纯化的rhCygb免疫BALB/c小鼠,采用甲基纤维素半固体培养基法获得抗rhCygb的单克隆抗体杂交瘤细胞,间接ELISA法筛选制备单克隆抗体,建立双抗体夹心ELISA法。结果筛选获取了稳定分泌单克隆抗体的杂交瘤细胞株,通过抗原表位相加法实验获得5株表位不同的细胞株,Western-blotting验证能与rhCygb特异性结合,间接ELISA法验证其不与本实验室制备的其它PET28a-BL21蛋白及BL21裂解液发生交叉反应。本方法灵敏度为1.25ng/ml,在浓度为10～1250ng/ml时,线性关系良好,相关性达0.9931,实验内和实验间平均变异系数分别为6.2%和10.92%。结论成功建立了灵敏度好、特异性高的双抗体夹心法,为下一步研究rhCygb药代动力学奠定了基础。     

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230

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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。     

抗磺胺嘧啶单克隆抗体的制备及其ELISA检测试剂盒的建立

目的 :制备抗磺胺嘧啶 (SD)的单克隆抗体 (mAb) ,并研制SD快速检测试剂盒。方法 :以SD BSA的偶联物作为抗原免疫BALB/c小鼠 ,采用杂交瘤技术制备抗SD的mAb。并用抗SDmAb和SD HRP酶标抗原 ,建立一种可检测样品中的游离SD分子的竞争法ELISA。结果 :获得 5株可分泌抗SDmAb的杂交瘤细胞 1A1、1B8、1E4、2C1和 3D9。其中 1A1、1B8和 1E4为IgG1,2C1和 3D9为IgG2。试剂盒的半数抑制率 (I5 0 )为 9.3μg/L ,理论最低检出量达到 0 .6μg/L ,对于不同样品中SD的回收率均高于 60 %。除与SD反应以外 ,该试剂盒对其他磺胺类药物检测的交叉反应均小于 3 %。结论 :成功地制备了抗SD的mAb ,并建立了一种可快速检测各种样品中SD的竞争ELISA试剂盒     

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.     

Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay(ELISA).Therefore,an anti-GP73 antibody with high specificity was highly demanded.In the present study,by hybridoma screening,we generated an anti-GP73 monoclonal antibody(mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression.The specificity of 6A2 was evaluated by Western blotting,immunohistochemistry and immunoprecipitation.The results showed that 6A2 recognized GP73 in both native and denatured forms.In addition,we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures,and measured the serum GP73 level of patients using this assay.Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls(P = 0.0036).Furthermore,for the first time,GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls(P = 0.0172).     

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.     

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.     

检测肌糖蛋白C夹心ELISA方法的建立及初步应用

目的:以已经制备的抗肌糖蛋白C(TN-C)的单克隆抗体(mAb)为基础,建立能定量检测肌糖蛋白C浓度的夹心ELISA方法,并初步于临床血清标本检测.方法:将3株mAb(克隆号分别为:1A8、3H7和4D6)制备腹水后纯化,分别与辣根过氧化物酶交联后,两两配对,以重组肌糖蛋白C 蛋白为检测抗原,分析抗体之间最佳组合;利用建立的夹心ELISA方法检测收集的临床骨肉瘤患者和正常人血清标本.结果:包被1A8 mAb,HRP-4D6配对敏感性最高;骨肉瘤患者血清中肌糖蛋白C浓度明显高于正常人.结论:成功建立检测肌糖蛋白C的夹心ELISA方法,并利用其检测到肌糖蛋白C在骨肉瘤患者中的浓度异于正常人.