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Home / Equine Research Bank / Vet Immunol Immunopathol / Generation and characterization of monoclonal antibodies to ...
Veterinary immunology and immunopathology2012; 147(1-2); 60-68; doi: 10.1016/j.vetimm.2012.04.003

Generation and characterization of monoclonal antibodies to equine NKp46.

Abstract: The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.
Copyright © 2012 Elsevier B.V. All rights reserved.
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Publication Date: 2012-04-07 PubMed ID: 22551980PubMed Central: PMC3354985DOI: 10.1016/j.vetimm.2012.04.003Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research involves the generation and testing of monoclonal antibodies that identify equine NKp46, a marker of NK cells in the horse’s immune system. The research will aid in a better understanding of the equine NK cell population which is largely unexplored.

Methodology

  • The research involved expressing the extracellular region of equine NKp46 with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46). This was used to generate mouse monoclonal antibodies (mAbs).
  • The generated mAbs were first screened using an Enzyme-Linked Immunosorbent Assay (ELISA) to test their ability to recognize NKp46 and to ensure they didn’t recognize IL-4 or the similar immunoreceptor CD16.
  • Nine mAbs which passed this screening were selected and tested for their ability to identify full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein.

Findings

  • The selected mAbs were found to recognize a population of lymphocytes, that were morphologically similar to NKp46+ cells in humans and cattle, via flow cytometric analysis.
  • Using representative mAb 4F2 in a study on nine horses, the researchers found that 0.8-2.1% of PBL were labelled, correlating with mean fluorescence intensity consistent with gene expression data.
  • MAb 4F2+ PBL were enriched by magnetic cell sorting and were observed to express higher levels of NKp46 mRNA than 4F2- cells, as determined by quantitative RT-PCR.
  • PBL from five horses that had CD3 removed contained more 4F2+ cells than unsorted PBL.
  • Through another ELISA test, researchers discovered that the nine mAbs recognized three different epitopes, indicating that they can be used to identify various forms of the NKp46 antigen.

Conclusion

  • The generated and characterized monoclonal antibodies to equine NKp46 would provide better tools to understand the largely uncharacterized equine NK cell population.

Cite This Article

APA
Noronha LE, Harman RM, Wagner B, Antczak DF. (2012). Generation and characterization of monoclonal antibodies to equine NKp46. Vet Immunol Immunopathol, 147(1-2), 60-68. https://doi.org/10.1016/j.vetimm.2012.04.003

Publication

Veterinary immunology and immunopathology
ISSN: 1873-2534
NlmUniqueID: 8002006
Country: Netherlands
Language: English
Volume: 147
Issue: 1-2
Pages: 60-68

Researcher Affiliations

Noronha, Leela E
  • Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States.
Harman, Rebecca M
    Wagner, Bettina
      Antczak, Douglas F

        MeSH Terms

        • Animals
        • Antibodies, Monoclonal / biosynthesis
        • Antibodies, Monoclonal / immunology
        • CHO Cells
        • Cricetinae
        • Cricetulus
        • Epitopes
        • Flow Cytometry
        • Horses / immunology
        • Interleukin-4 / genetics
        • Mice
        • Natural Cytotoxicity Triggering Receptor 1 / analysis
        • Natural Cytotoxicity Triggering Receptor 1 / genetics
        • Natural Cytotoxicity Triggering Receptor 1 / immunology
        • Recombinant Fusion Proteins / biosynthesis

        Grant Funding

        • F32 HD055794 / NICHD NIH HHS
        • R01 HD049545 / NICHD NIH HHS
        • F32 HD 055794 / NICHD NIH HHS

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        Citations

        This article has been cited 7 times.
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