Production and characterization of monoclonal antibodies against major histocompatibility complex class I chain-related gene A

Authors

  • W. Wongsena,

    1. PhD Program in Biomedical Science, Graduate School of Khon Kaen University, Khon Kaen, Thailand
    2. The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
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  • G. Sconocchia,

    1. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA
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    • Present address: Institute for Surgical Research and Hospital Management, University of Basel, Basel, Switzerland

  • H. S. Cho,

    1. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA
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  • C.-C. Chang,

    1. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA
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  • X. Wang,

    1. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA
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  • K. Klumkrathok,

    1. The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
    2. PhD Program in Medical Technology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand
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  • S. Ferrone,

    1. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA
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    •  

      Present address: Hillman Cancer Research Institute, University of Pittsburgh, Pittsburgh, PA, USA

  • C. Leelayuwat

    1. The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
    2. Department of Clinical Immunology and Transfusion Medicine, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
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Chanvit Leelayuwat, PhD
Department of Clinical Immunology and Transfusion Medicine
The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL)
Faculty of Associated Medical Sciences
Khon Kaen University
Khon Kaen 40002
Thailand
Tel/Fax: +66 4 320 2089
e-mail: chanvit@kku.ac.th

Abstract

Major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand for the activating immunoreceptor natural killer group 2D (NKG2D), is expressed on stressed cells such as tumor cells. Study of expression of this molecule on tumor cells and patients’ sera is useful to define patients’ stages leading to proper selection of therapy. In this study, mouse anti-MICA monoclonal antibodies (mAbs) were produced by DNA immunization using a gene gun. Screening of anti-MICA-producing mouse and hybridomas were performed by immunoblot and cell enzyme-linked immunosorbent assay (ELISA) against MICA-positive HeLa and -negative Me1386 cell lines. MAbs were characterized against MICA-positive and -negative cell lines by immunoblot, cell ELISA and flow cytometry. The mAbs were also characterized for locus and allele specificities of MICA and MHC class I chain-related gene B (MICB) as well as for their ability to stain formalin-fixed paraffin-embedded tissues by immunohistochemistry. Although all mouse immune sera were positive with MICA-positive cells by both immunoblot and cell ELISA methods, some hybridomas were positive only with one method. The mAbs had diverse specificities to detect MICA and MICB and different abilities to stain formalin-fixed paraffin-embedded tissues. Thus, DNA immunization by gene gun is an effective method to generate immune mice for the production of mAbs with a variety of specificities against native and denatured forms of MIC proteins.

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