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

Abstract

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

CLC Number:

S852.65^＋5

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.

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Preparation of Monoclonal Antibody Against Canine Rotavirus and Development of Colloidal Gold Strip Detection Method

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