犬轮状病毒单克隆抗体制备及胶体金试纸条检测方法的建立

doi:10.16431/j.cnki.1671-7236.2022.11.034

摘要/Abstract

摘要： 【目的】研发犬轮状病毒（Canine ratavirus，CRV）免疫学诊断试剂，制备并鉴定CRV特异性单克隆抗体，建立可用于检测CRV的胶体金免疫层析法。【方法】以CRV临床分离株SL006株为免疫原免疫BALB/c雌性小鼠，用杂交瘤细胞法进行细胞融合，通过间接免疫荧光法（IFA）筛选可稳定分泌CRV单克隆抗体的杂交瘤细胞，并制备腹水，亲和层析法进行纯化获得单克隆抗体。对获得的单克隆抗体进行鉴定，经条件优化建立胶体金试纸条检测方法，对试纸条的灵敏度、特异性、重复性和应用情况进行评价。【结果】获得了4株杂交瘤细胞，分别命名为2C12、4A5、1H3、5F3，IFA效价分别为1：6 400、1：12 800、1：1 600和1：3 200；重链亚类分别为IgG2b、IgG2a、IgG1和IgG2a，轻链亚类均为kappa；制备的胶体金试纸条检测线包被单克隆抗体4A5，对照线包被羊抗鼠IgG，金标垫包被胶体金标记的单克隆抗体2C12，对CRV病毒液的检测灵敏度为10^4.2 TCID₅₀/mL，检测犬源其他病毒犬细小病毒（Canine parvovirus，CPV）、犬副流感病毒（Canine parainfluenza virus，CPIV）、犬腺病毒Ⅰ型（Canine adenovirus-Ⅰ，CAV-Ⅰ）、CAV-Ⅱ、犬瘟热病毒（Canine distemper virus，CDV）病毒液及CRV阴性肛拭子和阴性粪便均为阴性；批内和批间重复性良好；利用制备的胶体金试纸条和RT-PCR方法对47份样品进行检测比较，两者符合率为93.6%。【结论】本研究建立的CRV胶体金试纸条检测方法具有良好的敏感性、特异性、重复性，与RT-PCR方法符合率较高，可为CRV的临床快速诊断提供有效的检测方法。

关键词: 犬轮状病毒(CRV); 单克隆抗体; 胶体金试纸条

Abstract: 【Objective】 This study was aimed to develop an immunological diagnostic reagent for Canine rotavirus (CRV), prepare and identify specific monoclonal antibodies (MAbs) against CRV, and establish colloidal gold immunochromatography for detection of CRV.【Method】 Female BALB/c mice were immunized with clinical CRV strain SL006, and cell fusion was performed using hybridoma cell method.Hybridoma cells that secreted CRV MAb were screened by indirect immunofluorescence assay (IFA).Then ascites were prepared and purified by affinity chromatography.The monoclonal antibodies obtained was identified and the colloidal gold strip detection method was established through optimization.The sensitivity, specificity, repeatability and application of the strip were evaluated respectively.【Result】 Four hybridoma cells were obtained, named 2C12, 4A5, 1H3 and 5F3, and the IFA titers were 1:6 400, 1:12 800, 1:1 600 and 1:3 200, respectively.Their heavy chains belonged to IgG2b, IgG2a, IgG1, IgG2a, and all light chains were kappa. The 4A5 was coated on a porous nitrocellulose membrane as the detection line, the goat anti-mouse IgG antibody was coated as the control line, and the MAb 2C12 was conjugated with colloid gold, respectively.The detection limit of the strip was 10^4.2 TCID₅₀/mL.Other viruses of canine origin, Canine parvovirus (CPV), Canine parainfluenza virus (CPIV), Canine adenovirus type Ⅰ (CAV-Ⅰ), CAV-Ⅱ, Canine distemper virus (CDV), CRV negative anal swabs and negative feces were negative.Intra-assay and inter-assay repeatability test results were consistent.47 samples were detected by the colloidal gold strip and RT-PCR, and the coincidence rate was 93.6%.【Conclusion】 The strip had high sensitivity and specificity, good repeatability, and high coincidence rate with RT-PCR method, providing an effective method for rapid clinical diagnosis of CRV.

Key words: Canine ratavirus (CRV); monoclonal antibodies; colloidal gold strip

中图分类号:

S852.65^＋5

赵少若, 黄甜, 郝丽影, 吴洪超, 汪志艳, 崔宁宁, 王亚玲, 邓均华, 田克恭. 犬轮状病毒单克隆抗体制备及胶体金试纸条检测方法的建立[J]. 中国畜牧兽医, 2022, 49(11): 4429-4437.

ZHAO Shaoruo, HUANG Tian, HAO Liying, WU Hongchao, WANG Zhiyan, CUI Ningning, WANG Yaling, DENG Junhua, TIAN Kegong. Preparation of Monoclonal Antibody Against Canine Rotavirus and Development of Colloidal Gold Strip Detection Method[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4429-4437.

参考文献

[1] 李六金, 夏咸柱, 武德丽, 等.犬轮状病毒的分离与鉴定[J].中国兽医学报, 1984, 4(1):26-32. LI L J, XIA X Z, WU D L, et al.Isolation and identification of Canine rotavirus[J]. Chinese Journal of Veterinary Science, 1984, 4(1):26-32.(in Chinese)
[2] 姜智礼.犬轮状病毒病的诊治[J].中国畜牧兽医文摘, 2016, 10:200. JIANG Z L.Diagnosis and treatment of Canine rotavirus disease[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine, 2016, 10:200.(in Chinese)
[3] KANG B K, SONG D S, JUNG K I, et al.Genetic characterization of Canine rotavirus isolated from a puppy in Korea and experimental reproduction of disease[J].Journal of Veterinary Diagnostic Investigation, 2007, 19(1):78-83.
[4] 高得仪.犬猫疾病学[M].北京:中国农业大学出版社, 2001. GAO D Y.Dog and Cat Diseases[M].Beingjing:China Agricultural University Press, 2001.(in Chinese)
[5] YAN N, TANG C, KAN R, et al.Genome analysis of a G9P
[23] group A Rotavirus isolated from a dog with diarrhea in China[J].Infection, Genetics and Evolution, 2019, 70:67-71.
[6] 陆江, 朱道仙, 刘静, 等.轮状病毒患犬与健康犬肠黏膜通透性及肠道菌群的差异性分析[J].扬州大学学报(农业与生命科学版), 2020, 41(1):21-26. LU J, ZHU D X, LIU J, et al.Diffference analysis of intestinal mucosal permeability and intestinal flora between Rotavirus infected dogs and healthy dogs[J].Journal of Yangzhou University (Agricultural and Life Science Edition), 2020, 41(1):21-26.(in Chinese)
[7] 皮泉, 熊力.规模养殖场疫病控制技术系列丛书犬病[M].贵阳:贵州科技出版社, 2017. PI Q, XIONG L.Scale Farm Disease Control Technology Series of Books Dog Disease[M].Guiyang:Guizhou Science and Technology Press, 2017.(in Chinese)
[8] ORTEGA A F, MARTÍNEZ-CASTAÑEDA J S, BAUTISTA-GÓMEZ L G, et al.Identification of co-infection by Rotavirus and Parvovirus in dogs with gastroenteritis in Mexico[J].Brazilian Journal of Microbiology, 2017, 48(4):769-773.
[9] LI Z, ZHAO F, TANG T, et al.Development of a colloidal gold immunochromatographic strip assay for rapid detection of Bovine rotavirus[J].Viral Immunology, 2019, 32(9):393-401.
[10] JIANG N, SHI L, LIN J, et al.Comparison of two different combined test strips with fluorescent microspheres or colored microspheres as tracers for Rotavirus and Adenovirus detection[J].Virology Journal, 2018, 15(1):44.
[11] 周梦婕, 李小盼, 代荣阳, 等.免疫层析试纸条检测技术的研究进展[J].检验医学与临床, 2019, 16(22):3382-3386. ZHOU M J, LI X P, DAI R Y, et al.Research progress of immunochromatographic strip detection technology[J]. Laboratory Medicine and Clinic, 2019, 16(22):3382-3386.(in Chinese)
[12] 武晋慧, 孟利.免疫胶体金技术及其应用研究进展[J].中国农学通报, 2019, 35(13):146-151. WU J H, MENG L.Immunocolloidal gold technology:Advances and application[J].Chinese Agricultural Science Bulletin, 2019, 35(13):146-151.(in Chinese)
[13] HARLOW E, LANE D.Antibodies:A Laboratory Manual[M].New York:Cold Spring Harbor Laboratory Press, 1998.
[14] 魏锁成, 何丽.犬轮状病毒荧光定量RT-PCR检测方法的建立[J].中国动物检疫, 2009, 26(5):38-41. WEI S C, HE L.Construction of Real-time fluorescence quantitative RT-PCR method for detecting Canine rotavirus[J].China Animal Health Inspection, 2009, 26(5):38-41.(in Chinese)
[15] PAPP H, MIHALOV-KOVÁCS E, DÓRÓ R, et al.Full-genome sequencing of a Hungarian canine G3P
[3] rotavirus A strain reveals high genetic relatedness with a historic Italian human strain[J].Virus Genes, 2015, 50(2):310-315.
[16] 赵远良, 岳城, 丑武江.犬病鉴别诊断与防治[M].北京:金盾出版社, 2008. ZHAO Y L, YUE C, CHOU W J.Differential Diagnosis and Prevention of Canine Diseases[M].Beijing:Golden Shield Press, 2008.(in Chinese)
[17] 魏锁成, 何丽.ELISA双抗体夹心法检测幼犬轮状病毒的研究[J].中国动物检疫, 2008, 25(1):37-38. WEI S C, HE L.Research on detection of puppy Rotavirus by double antibody sandwich ELISA[J].China Animal Health Inspection, 2008, 25(1):37-38.(in Chinese)
[18] WANG H, LIU M, SUGATA K, et al.Development of a new enzyme immunoassay for improved detection of Rotavirus in fecal specimens of vaccinated infants[J].Journal of Clinical Virology, 2018, 99-100:44-49.
[19] MOSALLANEJAD B, SHAPOURI M R S, AVIZEH R, et al.Antigenic detection of Canine rotavirus group A in diarrheic dogs in Ahvaz district, Southwestern Iran[J]. Comparative Clinical Pathology, 2015, 24(4):899-902.
[20] MOUTELÍKOVÁ R, DVOŘÁKOVÁ HEROLDOVÁ M, HOLÁ V, et al.Human rotavirus A detection:Comparison of enzymatic immunoassay and rapid chromatographic test with two quantitative RT-PCR assays[J].Epidemiologie Mikrobiologie Imunologie, 2018, 67(3):110-113.
[21] ELSCHNER M, PRUDLO J, HOTZEL H, et al.Nested reverse transcriptase-polymerase chain reaction for the detection of group A Rotaviruses[J].Journal of Veterinary Medicine, 2002, 49(2):77-81.
[22] LI C, LUO G, ZENG Y, et al.Establishment of sandwich ELISA for quality control in Rotavirus vaccine production[J].Vaccines (Basel), 2022, 10(2):243.
[23] CHAROENKUL K, JANETANAKIT T, BUNPAPONG N, et al.Molecular characterization identifies intra-host recombination and zoonotic potential of Canine rotavirus among dogs from Thailand[J].Transboundary and Emerging Diseases, 2021, 68(3):1240-1252.
[24] ARIAS C F, ROMERO P, ALVAREZ V, et al.Trypsin activation pathway of Rotavirus infectivity[J].Journal of Virology, 1996, 70(9):5832-5839.
[25] OTTO P H, ROSENHAIN S, ELSCHNER M C, et al.Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of Bovine species A rotavirus strains[J].Veterinary Microbiology, 2015, 179(3-4):168-176.
[26] 尹梅.猪瘟病毒抗体胶体金免疫层析快速检测试纸条的研制及初步应用[D].南京:南京农业大学, 2013. YIN M.Preparation and application of colloidal gold immunochromatography strip for CSFV antibody[D].Nanjing:Nanjing Agricultural University, 2013.(in Chinese)