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| 定量检测猪繁殖与呼吸综合征病毒游离受体夹心ELISA方法的建立与应用 | |
| 引用本文： | 夏文龙,吴植,郭长明,朱善元,余树培,张鑫宇,夏晓莉,孙怀昌.定量检测猪繁殖与呼吸综合征病毒游离受体夹心ELISA方法的建立与应用[J].中国畜牧兽医,2018,45(4):881-887. |
| 作者姓名： | 夏文龙 吴植 郭长明 朱善元 余树培 张鑫宇 夏晓莉 孙怀昌 |
| 作者单位： | 1. 扬州大学兽医学院, 江苏省重要动物疫病与人兽共患病协同创新中心, 扬州 225009; 2. 江苏农牧科技职业学院, 江苏省兽用生物制药高技术研究重点实验室, 泰州 225300 |
| 基金项目： | 江苏高校优势学科建设工程项目（PAPD）；江苏省农业科技自主创新资金项目CX （14）2087；江苏省自然科学基金（BK20151576）；江苏农牧科技职业学院重点支持项目（NSFPT201631） |
| 摘 要： | 为了建立猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）游离受体定量检测方法，本试验用表达猪唾液酸黏附素（Sn）游离受体的重组腺病毒rAd-Sn4D-Fc感染PK-15细胞，以细胞培养上清纯化的Sn4D-Fc游离受体作为标准抗原，鼠抗猪Sn免疫血清为一抗，生物素标记兔抗猪Sn多克隆抗体为二抗，建立定量检测Sn4D-Fc游离受体的双抗体夹心ELISA方法；用rAd-Sn4D-Fc注射仔猪，定期采集血清进行游离受体检测。结果显示，纯化Sn4D-Fc标准抗原的纯度为94.3%，能被Sn免疫血清识别；一抗的最佳工作浓度为5 μg/mL蛋白，二抗的最佳稀释度为1：4 000，夹心ELISA检测标准抗原的灵敏度为0.4 ng/mL，对照抗原无交叉反应；夹心ELISA能从重组腺病毒注射猪血清中检测到Sn4D-Fc游离受体，最高表达量为6.54 ng/mL，持续时间为15 d。研究结果表明，建立的双抗体夹心ELISA可用于PRRSV游离受体的体内外定量检测。 |
| 关 键 词： | 猪繁殖与呼吸综合征病毒(PRRSV) 游离受体 夹心ELISA 定量检测 |
Establishment and Application of Sandwich ELISA for Quantitative Detection of Porcine Reproductive and Respiratory Syndrome Virus Soluble Receptors |
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| XIA Wenlong,WU Zhi,GUO Changming,ZHU Shanyuan,YU Shupei,ZHANG Xinyu,XIA Xiaoli,SUN Huaichang.Establishment and Application of Sandwich ELISA for Quantitative Detection of Porcine Reproductive and Respiratory Syndrome Virus Soluble Receptors[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):881-887. | |
| Authors: | XIA Wenlong WU Zhi GUO Changming ZHU Shanyuan YU Shupei ZHANG Xinyu XIA Xiaoli SUN Huaichang |
| Institution: | 1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; 2. Jiangsu Key Laboratory for High-tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Animal Husbandry and Veterinary College, Taizhou 225300, China |
| Abstract: | To establish a sandwich ELISA for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) soluble receptors,porcine sialoadhesin (Sn) soluble receptor Sn4D-Fc was purified from the recombinant adenovirus-transduced PK-15 cell and used as the standard antigen,mouse anti-porcine Sn serum was used as the first antibody and biotin-labeled rabbit anti-porcine Sn polyclonal antibody as the second antibody.The established sandwich ELISA was validated by detection of the soluble receptor in the serum samples of rAd-Sn4D-Fc-injected pigs.The results showed that the Sn4D-Fc standard antigen was purified to a purity of 94.3%,and was able to be recognized by anti-Sn serum.By using 5 μg/mL of the first antibody and 1:4 000 of second antibody,the detection limit of established sandwich ELISA was up to 0.4 ng/mL without cross reaction to control antigen.The Sn4D-Fc soluble receptor could be detected in the rAd-injected pigs with the highest expression level of 6.54 ng/mL and the longest duration of 15 d.These data suggested that the established sandwich ELISA was usable for quantitative detection of PRRSV soluble receptors in vitro and in vivo. |
| Keywords: | porcine reproductive and respiratory syndrome virus (PRRSV) soluble receptor sandwich ELISA quantitative detection |
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