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KMID : 0390320110210020071
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Chungbuk Medical Journal
2011 Volume.21 No. 2 p.71 ~ p.82
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A novel monoclonal antibody (22C6) against CD34 established by alternate immunization
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Kim So-Seul
Hong Kwon-Pyo
Lee Kyoung-Mee
Lee Ok-Jun
Ji Gil-Yong
Song Hyung-Geun
Yoon Sang-Soon
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Abstract
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Purpose: We developed a monoclonal antibody (mAb) against CD34 established by alternate immunization of CD34 expressing leukemic cell lines.
Materials and Methods : A 22C6 mAb was developed from alternate immunization of KG1(acute myelogenous leukemia) and Molm-1(megakaryoblast) leukemic cell lines. Using flow cytometric analysis of normal peripheral blood cells, TF-1(CD34 positive cell line) and various T, B cell lines, we characterized 22C6 mAb. The molecular weight of 22C6 antigen was analyzed by immunoprecipitation. Immunohistochemistry of frozen tonsil was done. For epitope identification, we performed enzyme assay and blocking assay. We performed flow cytometric analysis of bone marrow cells of AML patient.
Results: We established a mAb 22C6 revealing TF-1 positive and normal PBMCs negative immunoreactivity by alternate immunization method. A 22C6 mAb revealed positive immunoreactivity on KG1, Molm-1 and TF-1, and negative immunoreactivity on normal PBMCs and T and B leukemic cell lines. Antigen immunoprecipitated by 22C6mAb showed approximately 100 kDa sized band under non-reducing condition. Immunohistochemical staining disclosed strong immunoreactivity on high endothelial venule. Using enzyme assay and blocking assay, we confirmed that 22C6mAb recognize CD34 class Ⅲ epitope. CD34 positive cells in bone marrow of AML patient were detected by 22C6 mAb and by a known CD34 mAb(27H2) with similar immunoreactivity.
Conclusion: A novel monoclonal antibody(22C6) against CD34 was developed by alternate immunization. Alternate immunization and screening method are a useful approaches for development of specific mAb against restrictedly-expressed cell surface antigen.
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KEYWORD
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Alternate immunization, Monoclonal antibody, Restrictedly expressed antigen, CD34, Epitope
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