猪德尔塔冠状病毒N蛋白单克隆抗体制备及双抗体夹心ELISA方法建立

doi:10.16431/j.cnki.1671-7236.2025.11.028

摘要/Abstract

摘要： 【目的】制备猪德尔塔冠状病毒(Porcine deltacoronavirus，PDCoV)N蛋白的单克隆抗体并建立一种检测PDCoV抗原的双抗体夹心ELISA方法，为猪群PDCoV感染情况的临床快速、准确检测提供技术支持。【方法】通过原核表达系统表达PDCoV N蛋白并验证，用其免疫BALB/c雌鼠，利用杂交瘤细胞融合技术筛选制备抗PDCoV N蛋白的特异性单克隆抗体，并通过Western blotting、间接免疫荧光试验(IFA)验证单克隆抗体的反应性和特异性。将筛选到的单克隆抗体进行辣根过氧化物酶(HRP)标记，并分别与未标记的单克隆抗体两两组合配对，选择最优抗体用于双抗体夹心ELISA检测方法的建立，通过棋盘法优化捕获抗体包被浓度和酶标抗体稀释度等条件，条件优化后通过梯度稀释法确定该方法的PDCoV N蛋白和PDCoV的检出限，并验证其重复性和特异性，最终检测临床样品验证该方法与反转录实时荧光定量PCR的一致性。【结果】成功表达出纯度较高的PDCoV N蛋白，用其免疫小鼠4次后其血清抗体效价达到1∶256 000，通过杂交瘤融合技术成功筛选到6株能稳定分泌单克隆抗体的杂交瘤细胞株(5D2-1、5D2-2、6G7-1、6G7-2、3C5和6F10)。Western blotting、IFA结果显示，筛选到的6株单克隆抗体可与PDCoV N蛋白特异性反应。配对试验结果表明，最佳捕获抗体为5D2-1，最佳检测抗体为6G7-1-HRP。本研究建立的双抗体夹心ELISA方法对PDCoV N蛋白的检出限为2 ng/mL，全病毒的检出限为7.89×10³ TCID₅₀/mL，具有良好的灵敏性。批间和批内重复试验变异系数均＜10%，且未见与其他常见猪肠道病毒发生反应，具有良好的重复性和特异性。临床样本检测结果显示，建立的双抗体夹心ELISA方法与反转录实时荧光定量PCR方法符合率为88.19%，Kappa值为0.761，表明该方法可用于PDCoV临床样品的检测。【结论】本研究成功筛选制备了6株针对PDCoV N蛋白的特异性单克隆抗体，建立了一种特异性好、灵敏度高的PDCoV双抗体夹心ELISA检测方法，为PDCoV临床诊断提供了新方法。

关键词: 猪德尔塔冠状病毒(PDCoV); N蛋白; 单克隆抗体; 双抗体夹心ELISA

Abstract: 【Objective】 The purpose of this study was to prepare monoclonal antibodies against Porcine deltacoronavirus (PDCoV) N protein and establish a double antibody sandwich ELISA method for detecting PDCoV antigen,so as to provide technical support for rapid and accurate clinical detection of PDCoV infection in pigs. 【Method】 PDCoV N protein was expressed by prokaryotic expression system,and BALB/c female mice were immunized with it.The specific monoclonal antibodies against PDCoV N protein were screened and prepared by hybridoma cell fusion technology,and the reactivity and specificity of monoclonal antibodies were verified by Western blotting and indirect immunofluorescence assay (IFA).The selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) and paired with unlabeled monoclonal antibodies,respectively.The optimal antibodies were selected for the establishment of a double antibody sandwich ELISA detection method.The conditions such as capture antibody coating concentration and enzyme-labeled antibody dilution were optimized by chessboard method.After optimization,the detection limits of PDCoV N protein and PDCoV of the method were determined by gradient dilution method,and their repeatability and specificity were verified.Finally,clinical samples were detected to verify the consistency of the method with reverse transcription Real-time quantitative PCR. 【Result】 PDCoV N protein was successfully expressed,and the serum antibody titer reached 1∶256 000 after 4 times of immunization.Six hybridoma cell lines (5D2-1,5D2-2,6G7-1,6G7-2,3C5 and 6F10) which could stably secrete monoclonal antibodies were successfully screened by hybridoma fusion technology.The results of Western blotting and IFA showed that the six selected monoclonal antibodies could specifically react with PDCoV N protein.The results of paired test showed that the best capture antibody was 5D2-1,and the best detection antibody was 6G7-1-HRP.The double antibody sandwich ELISA method established in this study had a detection limit of 2 ng/mL for PDCoV N protein and a detection limit of 7.89×10³TCID₅₀/mL for the whole virus,which had good sensitivity.The coefficients of variation of inter-assay and intra-assay repeated tests were ＜10%,and no reaction with other common porcine enteroviruses was observed,with good repeatability and specificity.The detection results of clinical samples showed that the coincidence rate between the double antibody sandwich ELISA method established in this study and the reverse transcription Real-time quantitative PCR method was 88.19%,and the Kappa value was 0.761,indicating that the method could be used for the detection of PDCoV clinical samples. 【Conclusion】 Six specific monoclonal antibodies against PDCoV N protein were successfully screened and prepared in this study,and a double antibody sandwich ELISA detection method for PDCoV with good specificity and high sensitivity was established,which provided a new method for clinical diagnosis of PDCoV.

Key words: Porcine deltacoronavirus (PDCoV); N protein; monoclonal antibody; double antibody sandwich ELISA

中图分类号:

S852.65⁺1

王田田, 于瑞明, 张莉萍, 白英杰, 张中旺, 潘丽, 张全伟, 刘新生. 猪德尔塔冠状病毒N蛋白单克隆抗体制备及双抗体夹心ELISA方法建立[J]. 中国畜牧兽医, 2025, 52(11): 5326-5337.

WANG Tiantian, YU Ruiming, ZHANG Liping, BAI Yingjie, ZHANG Zhongwang, PAN Li, ZHANG Quanwei, LIU Xinsheng. Preparation of Monoclonal Antibody Against N Protein of Porcine Deltacoronavirus and Establishment of Double Antibody Sandwich ELISA Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5326-5337.

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