ST细胞蛋白定量ELISA检测方法的建立及初步应用
发布时间：2018-08-25 10:39
【摘要】：猪睾丸细胞(Swine Testis, ST)细胞系于1960年由McClurkin AW等建立,属于成纤维细胞系,体外培养呈贴壁状态。该细胞系用于猪细小病毒(PPV)、伪狂犬病毒、(PRV)、猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)等的繁殖和猪瘟疫苗、猪细小病毒疫苗的制备,是病毒繁殖生产的重要细胞系之一。为了保证疫苗或生物制品的安全有效,对多种宿主细胞蛋白残留量进行了限定。因此,本研究对猪睾丸宿主细胞蛋白残留特异性检测方法进行了初步的研究。利用本实验室保存的猪睾丸细胞制备了细胞抗原,采用低剂量长程免疫制备多克隆抗血清。用间接ELISA和western blot方法对制备的抗血清效价和特异性进行了测定,结果表明,抗体效价大于1：10000；琼脂双扩散效价均大于1：4；制备的多克隆抗体具有良好的特异性,与DMEM、MEM、新生牛血清、胎牛血清、马血清均没有交叉反应。利用纯化的抗体,建立了猪睾丸宿主细胞蛋白定量检测的间接ELISA方法。所建立的方法具有良好的重复性、特异性和灵敏度,可以检测疫苗制品中大于25ng/mL的残留蛋白,标准曲线的线性范围为25-400ng/mL。对建立的间接ELISA定量检测方法进行了初步应用,对市售的猪瘟疫苗中猪睾丸细胞宿主蛋白残留量进行了检测。本研究建立了猪睾丸细胞蛋白残留量的间接ELISA定量检测方法,为生物制品中宿主细胞蛋白残留的控制提供了可靠的检测手段,为生物制品的安全有效提供了有力的保障。
[Abstract]:Porcine testicular (Swine Testis, ST) cell line was established by McClurkin AW et al in 1960 and belongs to fibroblast cell line. The cell line was used to reproduce porcine parvovirus (PPV), pseudorabies virus, (PRV), swine fever virus (CSFV), porcine transmissible gastroenteritis virus (TGEV) et al. The preparation of porcine parvovirus vaccine is one of the important cell lines for virus reproduction and production. In order to ensure the safety and effectiveness of vaccines or biological products, several host cell protein residues were limited. Therefore, the specific detection method of protein residues in pig testicular host cells was studied. Cell antigens were prepared from pig testicular cells preserved in our laboratory and polyclonal antisera were prepared by low dose long range immunization. The titer and specificity of the prepared antiserum were determined by indirect ELISA and western blot. The results showed that the titer of antibody was more than 1: 10000, the double diffusion titer of Agar was more than 1: 4. The polyclonal antibody prepared had good specificity. There was no cross reaction with DMEM,MEM, newborn bovine serum, fetal bovine serum and horse serum. Using purified antibody, an indirect ELISA method for quantitative detection of pig testicular host cell protein was established. The method has good reproducibility, specificity and sensitivity, and can be used to detect residual proteins larger than 25ng/mL in vaccine products. The linear range of the standard curve is 25-400ng / mL. The indirect ELISA quantitative detection method was applied to detect the residual protein of pig testicular cell host protein in swine fever vaccine. In this study, an indirect ELISA quantitative method for the detection of pig testicular protein residues was established, which provided a reliable method for the control of host cell protein residues in biological products, and provided a strong guarantee for the safety and effectiveness of biological products.
【学位授予单位】：西北民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：2202631
[Abstract]:Porcine testicular (Swine Testis, ST) cell line was established by McClurkin AW et al in 1960 and belongs to fibroblast cell line. The cell line was used to reproduce porcine parvovirus (PPV), pseudorabies virus, (PRV), swine fever virus (CSFV), porcine transmissible gastroenteritis virus (TGEV) et al. The preparation of porcine parvovirus vaccine is one of the important cell lines for virus reproduction and production. In order to ensure the safety and effectiveness of vaccines or biological products, several host cell protein residues were limited. Therefore, the specific detection method of protein residues in pig testicular host cells was studied. Cell antigens were prepared from pig testicular cells preserved in our laboratory and polyclonal antisera were prepared by low dose long range immunization. The titer and specificity of the prepared antiserum were determined by indirect ELISA and western blot. The results showed that the titer of antibody was more than 1: 10000, the double diffusion titer of Agar was more than 1: 4. The polyclonal antibody prepared had good specificity. There was no cross reaction with DMEM,MEM, newborn bovine serum, fetal bovine serum and horse serum. Using purified antibody, an indirect ELISA method for quantitative detection of pig testicular host cell protein was established. The method has good reproducibility, specificity and sensitivity, and can be used to detect residual proteins larger than 25ng/mL in vaccine products. The linear range of the standard curve is 25-400ng / mL. The indirect ELISA quantitative detection method was applied to detect the residual protein of pig testicular cell host protein in swine fever vaccine. In this study, an indirect ELISA quantitative method for the detection of pig testicular protein residues was established, which provided a reliable method for the control of host cell protein residues in biological products, and provided a strong guarantee for the safety and effectiveness of biological products.
【学位授予单位】：西北民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
【参考文献】
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1 王辉;张月兰;过琴媛;贾丽丽;钟书声;赵玉秀;马可;罗书荣;;疫苗制品中Vero细胞残余蛋白含量检测方法的建立[J];中国生物制品学杂志;2007年12期
，本文编号：2202631
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