Preparation and specific identification of monoclonal antibodies targeting the VP2 structural protein of minute virus of canines

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods Pub Date : 2025-02-15 DOI:10.1016/j.jim.2025.113833

Xiang Ren , Zhiping Hei , Kai Ji , Jianhui Guo , Yan Yan , Yuning Sun

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引用次数: 0

Abstract

The Minute Virus of Canines (MVC), classified under the genus Bocaparvovirus, causes severe respiratory and gastrointestinal symptoms in neonatal canines worldwide. The structural protein VP2 is essential for the attachment, infection, uncoating, and induction of the host immunological response to MVC. This study aimed to prepare a monoclonal antibody (mAb) against VP2 using the hybridoma technique. AlphaFold and CavityPlus bioinformatics analysis revealed that the N-terminal region of VP2 (amino acids 1–300) possesses structural characteristics that make it the most suitable target for effective antibody generation. The recombinant plasmid pET-32a(+)-VP2(N300) with fused Trx and His tags was successfully constructed. After immunizing mice, nine hybridoma cell lines were obtained, namely 1G5, 1G5–1, 1I24, 1I24–1, 2E6–1, 2 N9, 3C12–1, 4 M1, and 4 M1–1. The ascitic antibody titers of all cell lines were above 1:100,000. Western blot analysis of Walter Reed canine cells infected with MVC indicated the selection of three strains of monoclonal antibodies with strong specificity: 1G5, 3C12–1, and 4 M1. These three strains can be employed in immunofluorescence (IF) and immunoprecipitation (IP) tests for detecting VP2 protein. The monoclonal antibody mAb VP2 prepared in this study may serve as a valuable tool for detecting MVC and beneficial for investigating the mechanisms of MVC infection.

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来源期刊

Journal of immunological methods 医学-免疫学

CiteScore

4.10

自引率

0.00%

发文量

120

审稿时长

3 months

期刊介绍： The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.