China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (11): 5326-5337.doi: 10.16431/j.cnki.1671-7236.2025.11.028

• Preventive Veterinary Medicine • Previous Articles    

Preparation of Monoclonal Antibody Against N Protein of Porcine Deltacoronavirus and Establishment of Double Antibody Sandwich ELISA Method

WANG Tiantian1, YU Ruiming1, ZHANG Liping2, BAI Yingjie2, ZHANG Zhongwang2, PAN Li2, ZHANG Quanwei1, LIU Xinsheng2   

  1. 1. College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China;
    2. State Key Laboratory of Animal Disease Prevention and Control, Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
  • Received:2025-03-01 Published:2025-10-30

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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

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