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

The Journal of Biomedical Research 2012年第6期26卷 467-473页

作者： Aixia Zhang Brian Cao Department of Pharmacology School of Pharmacy Nanjing Medical University Nanjing Jiangsu 210029 China Antibody Technology Laboratory Van Andel Research Institute Grand Rapids M149503 USA.

Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar 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 as- say （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 recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, 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）.

关键词： GP73 monoclonal antibody Western blotting sandwich elisa hepatocellular carcinoma

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Development and Preliminary Application of Double Antibody sandwich elisa for Detection of Swine Vesicular Disease Virus

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Animal Husbandry and Feed Science 2011年第4期3卷 24-26,42页

作者： WU Guo-hua YAN Xin-min ZHAO Zhi-xun CUI Li-fan LI Jian ZHU Hai-xia ZHU Cai-zhu ZHANG Qiang State Key Laboratory of Veterinary Etiological Biology National Foot and Mouth Disease Reference Laboratory Lanzhou Veterinary Research In-stitute Chinese Academy of Agricultural Sciences Lanzhou 730046 China

[Objective] The aim of this study was to develop an indirect sandwich EUSA method for detection of swine vesicular disease virus （SVDV）. [Method] High titer serum against SVDV was prepared respectively by inoculated rabbits and guinea pigs with purified virus. To develop an indirect sandwich elisa, the optimum concentrations of capture antibody, detection antibody, enzyme conjugate and standard antigen were determined using block titration, and positive threshold value was also determined. The specificity, sensitivity and repeatability of the developed Ielisa were evaluated using cross-reaction test, comparison test and intra-assay repeated test. In addition, standard samples and clinical samples were detected by this method. [ Result] The best working conditions of the developed elisa are as follows： capture antibody, 1：400; detection antibody, 1 ： 200; enzyme conjugate, 1 ： 8 000; and standard antigen, 1 ： 4. The positive threshold value was found to be 0.20. For the detection by the developed EUSA, no cross-reaction with foot and mouth disease was observed. The developed elisa had close sensitivity with elisa recommended by the World Organisation for Animal Health （OIE） but had sensitivity 2 -4 times higher than that of reverse indirect hernagglutination test. In addition, the developed method also had good reproducibility, and the detection results of standard samples were in line withtheir own background. All the 36 clinical samples were negative in the developed elisa. [ Conclusion] The developed indirect sandwich elisa can be used for diagnosis of swine vesicular disease.

关键词： Swine vesicular disease virus sandwich elisa Diagnosis

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Selection of specific nanobodies against peanut allergen through unbiased immunization strategy and the developed immuno-assay

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Food Science and Human Wellness 2023年第3期12卷 745-754页

作者： Yaozhong Hu Chuan Zhang Jing Lin Yi Wang Sihao Wu Ying Sun Bowei Zhang Huan Lv Xuemeng Ji Yang Lu Shuo Wang Tianjin Key Laboratory of Food Science and Health School of MedicineNankai UniversityTianjin 300071China College of Food Science and Engineering Tianjin University of Science and TechnologyTianjin 300457China

Peanut allergy is considered to be a major health issue with global *** date,no effective curative approach has been applied for the therapy of the anaphylaxis resulting from the peanut *** accurate and effective detection methods for the surveillance of allergens in food are still the primary strategies to avoid allergic *** this study,nanobodies(Nbs)derived from the Heavy-Chain only Antibodies(HCAbs)were selected against the general peanut protein extract through the unbiased strategy to facilitate the development of the sandwich elisa for the detection and surveillance of peanut allergen *** target antigen of the selected Nb was identified as peanut allergen Ara h 3,and a cross-reaction was observed with the member of Gly 1 from the Ara h 3 *** applicability of the self-paired Nb P43 on the establishment of the immuno-assay was verified.A sandwich elisa against peanut allergen was developed,which reached a linear range of 0.2-10.6μg/mL,and a limit of detection of 53.13 ng/mL.

关键词： Peanut allergen Ara h 3 Nanobody sandwich elisa Gly 1

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Detection of Human Epididymis Protein 4(HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based sandwich Enzyme-Linked Immunosorbent Assay(elisa)

Detection of Human Epididymis Protein 4(HE4) in Human Serum ...

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第九届全国免疫学学术大会

作者： Zhi-qiang Lv ZH Dun Wen-ji Zhang Yu-ming Zhang Shu-hong Luo Jian-ming Fang Ruo-Pan Huang South China Biochip Research Center

The human epididymis protein 4(HE4may have a higher specificity in detection of non-malignant ***,it is essential to develop an immunoassay to detect ***:In our study,a fusion gene was constructed encoded with the HE4 ***,the protein produced in the bacterial cells(***)was used to immunize mice in order to generate hybridomas specific to *** supernatants were screened and four positive anti-HE4 cell lines which were *** cell lines produce monoclonal antibodies to HE4 epitopes,as demonstrated in the western blot for the recognition of a specific band by HE4 antibody and direct *** the antibodies cultivated,we successfully developed several good pair ***,we measured HE4 levels in 60 clinical human serum samples using a new method incorporating ***:Compared with the FDA-approved kit(Roche),our results showed a strong positive correlation to those two ***:In summary,the high-sensitivity antibodies were screened against HE4,and a sandwich elisa was developed as an accurate and feasible analytical tool for rapid detection of HE4 in human serum samples,which is especially valuable for diagnosing ovarian carcinomas.

关键词： HE4 monoclonal antibody sandwich elisa ovarian cancer immunoassay

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Enrichment-elisa for Detection of Salmonella typhi From Food and Water Samples

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Biomedical and Environmental Sciences 2008年第2期21卷 137-143页

作者： S. KUMAR K. BALAKRISHNA HV. BATRA Division of Microbiology Defence R & D Establishment Jhansi Road Gwalior 474002 India

Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （selisa） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimization of enrichment procedures for use with the developed selisa to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin （H=d） antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An selisa was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the selisa, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water （BPW） and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed selisa with preceding enrichment step in BPW （Enrichment-elisa） was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food （30） and water （35） samples collected from field were also tested by Enrichment-elisa and culture method. Results Out of four specific clones to H=d antigen, one clone （# 2/56, IgG2a isotype） was used in selisa. The selisa had the detection limit of 10^4-10^5 cfu of S. typhi. Of the various broths used with selisa, BPW was found to yield maximum elisa values. Enrichment-elisa, when tested in artificially inoculated food samples, generally, could detect 10^2 S. typhi cfu/mL within 10 h from various food rinses （meat, vegetable） and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples （water） gave false positive result by Enr

关键词： Enrichment-elisa Food Monoclonal antibody Salmonella sandwich elisa Water

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抗磷脂酰乙醇胺结合蛋白1（PEBP1）的鼠单克隆抗体的制备及应用

抗磷脂酰乙醇胺结合蛋白1（PEBP1）的鼠单克隆抗体的制备及应用

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作者：王双双南京医科大学

学位级别：硕士

磷脂酰乙醇胺结合蛋白1(phosphatidylethanolamine binding protein 1)即PEBP1,也称Raf激酶抑制蛋白(raf kinase inhibitor protein, RKIP),属于磷脂酰乙醇胺结合蛋白家族,广泛存在于各种不同的生物中,参与了对细胞内多种信号转导通路... 详细信息

磷脂酰乙醇胺结合蛋白1(phosphatidylethanolamine binding protein 1)即PEBP1,也称Raf激酶抑制蛋白(raf kinase inhibitor protein, RKIP),属于磷脂酰乙醇胺结合蛋白家族,广泛存在于各种不同的生物中,参与了对细胞内多种信号转导通路的调节作用。PEBP1可以与Raf-1结合从而抑制MAPK信号转导通路,并参与了对G蛋白偶联受体信号通路和NF-κB信号通路的调控。PEBP1在膜的生物合成,精子发生,神经发育和细胞凋亡等生理过程中发挥重要作用。2003年Fu et al发现PEBP1可以抑制肿瘤细胞转移,使其成为抗肿瘤领域一个新的热点。目前一致被认为在前列腺癌,乳腺癌,结直肠癌,胃肠道间质瘤,黑色素瘤,卵巢上皮癌中具有抑制肿瘤转移的作用,并且是预后的标志。但是在胰腺癌中的表达以及作用还没有报道。本实验室在前期工作中已经获得大量高纯度的PEBP1蛋白标准品。本课题主要在于抗PEBP1的鼠单克隆抗体的制备及鉴定,双抗体夹心elisa方法的建立及研究其在胰腺癌组织中的应用。本课题的主要内容及设计如下:1.抗PEBP1抗体的制备及初步鉴定。2.双抗体夹心elisa方法的建立3.抗PEBP1抗体在胰腺癌组织中的应用。材料与方法1.抗PEBP1抗体的制备及初步鉴定:以纯化的PEBP1蛋白免疫BALB/c小鼠,之后取脾细胞使之与骨髓瘤细胞OUR-1融合。随后通过elisa筛选阳性杂交瘤,经过三轮的亚克隆,选择4个OD值最高,并且有不同活性的克隆,使用Protein G HP纯化获得抗体。获得抗体之后,进行抗体的初步鉴定,选择高表达和低表达该蛋白的人胰腺癌细胞系,通过Western Blot鉴定该组抗体识别该蛋白的特异性。将GFP-PEBP1转染到NIH3T3细胞中,将其固定,用所制备的抗体进行免疫荧光实验。2.双抗体夹心elisa方法的建立通过ForteBio’s Octet系统选择1对与该蛋白结合力高的抗体做sandwich elisa,可以用于进一步快速早期检测和诊断胰腺癌。3.抗PEBP1抗体在胰腺癌组织中的应用PEBP1被认为是抑制肿瘤转移的基因,我们在此将抗体用于组织芯片,研究PEBP1在胰腺癌病人和正常人中的表达是否不同。研究结果1.成功获得四株单克隆抗体,分别有不同的活性。7F12在Western Blot中可以识别正常的293细胞和部分胰腺癌细胞中的PEBP1.2.由ForteBio’s Octet系统筛选出一对抗体:4F10和8E2,可以识别PEBP1蛋白的不同的表位,因此这一对抗体可以用于sandwich elisa,它们可以检测出PEBP1在ng/ml的水平,经过优化之后可以用于临床实践中测定病人血清,可以进一步快速早期检测和诊断胰腺癌。3.单克隆抗体4A11在组织芯片中可以检测到在正常的胰腺组织中的PEBP1。而在胰腺癌组织中,PEBP1的表达很低甚至没有。结论我们的结果显示PEBP1在正常的胰腺中高表达,而在胰腺癌组织中的表达明显降低或者消失。抗PEBP1的单克隆抗体4A11, 4F10, 7F12和8E2是潜在的临床诊断胰腺癌的试剂。

关键词： PEBP1 单克隆抗体胰腺癌 sandwich elisa 组织芯片

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转基因外源蛋白EPSPS单克隆抗体制备及免疫分析方法建立

转基因外源蛋白EPSPS单克隆抗体制备及免疫分析方法建立

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中国棉花学会2013年年会

作者：谭桂玉曹振张亮张威南铁贵王保民中国农业大学农学与生物技术学院

转基因作物商品化进程中,转基因作物的安全评估是亟待解决的关键问题,建立准确、方便和快速的转基因外源蛋白鉴定和检测方法意义重大。本实验克隆epsps基因并构建epsps-pET30a表达载体,表达纯化EPSPS融合蛋白,以该蛋白为免疫原,制备兔... 详细信息

转基因作物商品化进程中,转基因作物的安全评估是亟待解决的关键问题,建立准确、方便和快速的转基因外源蛋白鉴定和检测方法意义重大。本实验克隆epsps基因并构建epsps-pET30a表达载体,表达纯化EPSPS融合蛋白,以该蛋白为免疫原,制备兔血清和小鼠单克隆抗体,进而进行棉花中转基因外源蛋白EPSPS检测方法(sandwich elisa)的建立和应用。实验结果表明:克隆得到1368bp的epsps基因,将其与pET-30a连接构建得到重组表达载体epsps-pET30a,诱导表达得到54kD的EPSPS融合蛋白,以该蛋白为免疫原制得抗兔血清,效价达到1.6×104。制得小鼠单克隆抗体4E3,效价为2×104,属于IgG1类,轻链为κ型。基于EPSPS兔血清和单克隆抗体的sandwich elisa检测范围为1～100ng·mL-1。往大豆、棉子和空白提取液中添加EPSPS蛋白,以建立的sandwichelisa测定添加回收率,分别为75.2%～90.8%、74.4%～78.4%和84.3%～87.3%,变异系数在1.36%～4.48%、1.20%～3.84%和0.92%～5.36%。交叉反应率结果表明,所建立方法与其他常用转基因外源蛋白(Cry1Ac、2Aa、9C、CpTI、PAT)没有交叉反应。用sandwich elisa测定10个棉花品种中EPSPS的含量。A1-1,A2-1,A3-1,A4-1和A5-1中检测到EPSPS蛋白,A1-2,A2-2,A3-2,A4-2和A5-2五个品种检测不到EPSPS。将所建立的EPSPS sandwich elisa同国外商品化检测试剂盒进行比较,本实验建立的方法与比较方法的结果一致,且本方法得到纯化的标准蛋白,还可进行定量测定。

关键词：转基因外源蛋白 EPSPS 单克隆抗体 sandwich elisa

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