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China Biotechnology
China Biotechnology  2022, Vol. 42 Issue (4): 17-23    DOI: 10.13523/j.cb.2110009
Screening of Monoclonal Antibodies Targeting the Equine IgG1 Based on Single B Cell Antibodies Gene Amplification Technology
CHEN Yang1,LIU Tong1,ZHANG Jia-qi2,LIAO Hua-xin1,LIN Yue-zhi2,WANG Xiao-jun2,***(),WANG Ya-yu1,***()
1 Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 5106321, China
2 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,the Chinese Academy of Agricultural Sciences, Harbin 150069, China
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In the field of equine immunology, research on equine B lymphocytes has been greatly hampered by the lack of commercial monoclonal antibodies of IgG. IgG is an important component of B cell receptor (BCR), which is associated with the differentiation and maturation of B cells. In order to obtain specific monoclonal antibodies of equine IgG, single B lymphocyte amplification was used to screen the antibodies. Firstly, the codon of equine IgG protein (EqIgG1-C) was optimized and synthesized on eukaryotic expression vector pcDNA3.4, and the antigenic protein was purified. Subsequently, the mice were immunized with the protein, and after the spleen cells were separated, the specific single B lymphocyte was separated by flow cytometry. The variable region genes of heavy and light chain of antibody were amplified by overlapping PCR method, and the complete antibody was identified. Finally, 27 strains of specific recombinant monoclonal antibodies were obtained from 80 B cells, and 3 strains with the strongest linear binding activity were selected and constructed into expression vector. After co-transfection of Expi293FTM cells, antibodies were expressed and purified. Verified by ELISA and Western blot, the results showed that the antibodies has extraordinary binding activity to EqIgG1-C protein. Using this method can save time and obtain specific antibodies efficiently, which provides an important research tool for the study of equine immunology.

Key wordsEquine IgG      Single B cell PCR      Antibody screening technology      Monoclonal antibody     
Received: 09 October 2021      Published: 05 May 2022
ZTFLH:  Q816  
Corresponding Authors: Xiao-jun WANG,Ya-yu WANG     E-mail:;
Cite this article:

CHEN Yang, LIU Tong, ZHANG Jia-qi, LIAO Hua-xin, LIN Yue-zhi, WANG Xiao-jun, WANG Ya-yu. Screening of Monoclonal Antibodies Targeting the Equine IgG1 Based on Single B Cell Antibodies Gene Amplification Technology. China Biotechnology, 2022, 42(4): 17-23.

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Fig.1 Schematic diagram of preparation of McAbs based on single B cell antibody technology
Fig.2 Construction strategy of immunogenic protein expression vector
Fig.3 Expression and identification of horse IgG1-C protein M: Marker;1, 3:EqIgG1-C nonreducing state;2, 4:EqIgG1-C reducing state; 1, 2 SDS-PAGE detection results;2, 4 Western blot detection results
Fig.4 Antibody titers test results of immunized mice
Fig.5 Sorting of EqIgG1-C protein specific memory B cells by flow cytometry
Fig.6 PCR amplification of heavy/light chain variable region (a) Heavy chain variable region (b)Kappa chain variable region
Fig.7 Expression and identification of EqIgG1-C specific mAb (a) Detection of 3 recombinant antibodies 1-3: IgG561, IgG563, IgG641(non-reduced); 4-6: IgG561, IgG563, IgG641(reduced) (b)Westen blot detection of 3 antibodies 1, 3, 5: EqIgG1-C reduced; 2, 4, 6: EqIgG1-C non-reduced; 1,2: Detected by IgG561; 3,4: Detected by IgG563; 5,6: Detected by IgG641 (c)Detection of different recombinant antibody titers
[1]   李克斌. 2017年生效的世界动物卫生组织疫病感染及侵染名录. 兽医导刊, 2017(3): 53-56.
[1]   Li K B. OIE-Listed dseases, infections and Infestations in 2017. Veterinary Orientation, 2017(3): 53-56.
[2]   Torres R M, Flaswinkel H, Reth M, et al. Aberrant B cell development and immune response in mice with a compromised BCR complex. Science, 1996, 272(5269): 1804-1808.
pmid: 8650582
[3]   Good-Jacobson K L, Shlomchik M J. Plasticity and heterogeneity in the generation of memory B cells and long-lived plasma cells: the influence of germinal center interactions and dynamics. Journal of Immunology (Baltimore, Md: 1950), 2010, 185(6): 3117-3125.
doi: 10.4049/jimmunol.1001155
[4]   Mcheyzer-Williams L J, Mcheyzer-Williams M G. Antigen-specific memory B cell development. Annual Review of Immunology, 2005, 23: 487-513.
doi: 10.1146/annurev.immunol.23.021704.115732
[5]   Lewis M J, Wagner B, Woof J M. The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies. Molecular Immunology, 2008, 45(3): 818-827.
doi: 10.1016/j.molimm.2007.06.158
[6]   Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256 (5517): 495-497.
doi: 10.1038/256495a0
[7]   Bradbury A R M, Sidhu S, Dübel S, et al. Beyond natural antibodies: the power of in vitro display technologies. Nature Biotechnology, 2011, 29 (3): 245-254.
doi: 10.1038/nbt.1791 pmid: 21390033
[8]   Hoogenboom H R. Selecting and screening recombinant antibody libraries. Nature Biotechnology, 2005, 23(9): 1105-1116.
pmid: 16151404
[9]   Liao H X, Levesque M C, Nagel A, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of Virological Methods, 2009, 158(1-2): 171-179.
doi: 10.1016/j.jviromet.2009.02.014
[10]   Liao H X, Lynch R, Zhou T, et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature, 2013, 496 (7446): 469-476.
doi: 10.1038/nature12053
[11]   Cao Y L, Su B, Guo X H, et al. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients B cells. Cell, 2020, 182(1): 73-84.e16.
doi: 10.1016/j.cell.2020.05.025
[12]   Whittle J R R, Wheatley A K, Wu L, et al. Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages. Journal of Virology, 2014, 88(8): 4047-4057.
doi: 10.1128/JVI.03422-13 pmid: 24501410
[13]   Glukhova X A, Prusakova O V, Trizna J A, et al. Updates on the production of therapeutic antibodies using human hybridoma technique. Current Pharmaceutical Design, 2016, 22(7): 870-878.
pmid: 26696411
[14]   Xu P, Ghosh S, Gul A R, et al. Screening of specific binding peptides using phage-display techniques and their biosensing applications. TrAC Trends in Analytical Chemistry, 2021, 137: 116229.
doi: 10.1016/j.trac.2021.116229
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