[1]贾宇旻,徐 飞,边洪芬,等.猪流行性腹泻病毒单克隆抗体的制备及胶体金免疫层析检测方法的建立[J].中国预防兽医学报,2019,(09):924-928.[doi:0.3969/j.issn.1008-0589.201901024]
 JIA Yu-min,XU Fei,BIAN Hong-fen,et al.Preparation of monoclonal antibodies against porcine epidemic diarrhea virus and establishment of colloidal gold immunochromatographic assay[J].Chinese journal of preventive veterinary medicine,2019,(09):924-928.[doi:0.3969/j.issn.1008-0589.201901024]
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猪流行性腹泻病毒单克隆抗体的制备及胶体金免疫层析检测方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年09
页码:
924-928
栏目:
诊断和检测技术
出版日期:
2019-10-25

文章信息/Info

Title:
Preparation of monoclonal antibodies against porcine epidemic diarrhea virus and establishment of colloidal gold immunochromatographic assay
文章编号:
1008-0589(2019)09-0924-05
作者:
 

贾宇旻12徐 飞3边洪芬3王 娟2贾爱卿2唐 勇3贾杏林1*

 (1. 湖南农业大学 动物医学院,湖南 长沙 410128;2. 广东海大畜牧兽医研究院有限公司,广东 广州 511400;
3. 暨南大学 生物工程学系/广东省抗体药物与免疫检测工程技术研究中心,广东 广州 510632)
Author(s):
 

JIA Yu-min12 XU Fei3 BIAN Hong-fen3 WANG Juan2 JIA Ai-qing2 TANG Yong3 JIA Xing-lin1*

 (1. College of veterinary medicine, Hunan Agricultural University, Changsha 410128, China; 2. Guangdong Haid Institute of Animal Husbandry & Veterinary, Guangzhou 511400, China; 3. Department of Bioengineering, Guangdong Province Engineering Research Center for Antibody Drug and Immunoassay, Jinan University, Guangzhou 510632, China)
关键词:
猪流行性腹泻病毒单克隆抗体表面荧光吸附胶体金免疫层析
Keywords:
  Porcine epidemic diarrhea virus  monoclonal antibody  cell surface fluorescence immunosorbent assay  colloidal gold immunochromatograohic assay
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201901024
文献标志码:
A
摘要:
为了建立快速检测猪流行性腹泻病毒(PEDV)的胶体金免疫层析方法(GICA),本研究用PEDV免疫BALB/c小鼠,制备杂交瘤细胞。分别采用间接ELISA和表面荧光吸附法(CSFIA)筛选,共得到33株能够稳定分泌抗PEDV单克隆抗体(MAb)的杂交瘤细胞株。通过制备小鼠腹水并纯化后测得其中21株细胞分泌的MAb的ELISA效价在1∶1.6×104以上,且均仅与PEDV反应,不与TGEV和PRV反应,特异性较强。采用双抗体夹心ELISA法筛选出能够配对检测PEDV的MAb有5株,以其中的4H7为检测抗体、5A9为俘获抗体初步建立了检测PEDV的GICA。该GICA特异性试验结果显示,除了PEDV检测结果为阳性外,该方法对伪狂犬病病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒和传染性胃肠炎病毒的检测结果均为阴性,特异性较强;该方法针对PEDV的最低检测限为7.8×103 TCID50/mL,敏感性较高;批内和批间重复性检测样品结果无明显差异,具有较好的重复性;将该方法和RT-PCR方法同时检测115份临床样品,该方法与RT-PCR阳性符合率为93 %。本实验制备的PEDV MAb,以及基于该AMbs初步建立的检测PEDV的GICA为开发快速检测PEDV的胶体金免疫层析试纸条奠定了基础。
  
Abstract:
To establish a colloidal gold immunochromatographic assay (GICA) for rapid detection of porcine epidemic diarrhea virus (PEDV), BALB/c mice were immunized with purified PEDV virion to prepare hybridoma cells. Thirty-three hybridoma cell lines that specific secreting anti-PEDV monoclonal antibody (MAb) were obtained after being screened by both indirect ELISA and cell surface fluorescence immunosorbent assay (CSFIA). The purified MAb titers of 21 cell lines were more than 1∶16,000 with high specificity by ELISA. Five strains of MAbs were found with capability of pairing for detection of PEDV by sandwich ELISA, and the clone 4H7 and 5A9 were selected as detection antibody and capture antibody, respectively, to develop the colloidal gold immunochromatographic assay against PEDV. The sensitivity, specificity, and repeatability of the GICA were evaluated and the results showed that negative reaction to antigens from pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV), which indicated that this method had high specificity. The minimum detection limit for detecting PEDV was 7.8×103 TCID50/mL suggested good sensitivity of the GICA. Repeated test samples with different batches of test strips showed no significant difference. 115 clinical samples were tested, and the coincidence rate with RT-PCR was 93%. Conclusion, the PEDV MAbs prepared in this study and the GICA based on these MAbs provide a basis for the development of colloidal gold test strips for rapid detection of PEDV.

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(本文编辑:李 娜;英文编辑:孟凡丹)

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更新日期/Last Update: 2019-10-29