Immune Netw.  2010 Dec;10(6):239-246. 10.4110/in.2010.10.6.239.

Characterization of a Novel Monoclonal Antibody (27H2) Recognizing Human CD34 Class III Epitope

• Hong KP
• Kang SH
• Lee KM
• Ji GY
• Yoon SS
• Kim JS
• Son BR
• Lee DG
• Lee OJ
• Song HG

• KMID: 2150680
• DOI: http://doi.org/10.4110/in.2010.10.6.239

Abstract

BACKGROUND
Monoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia.
METHODS
27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done.
RESULTS
Only KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity.
CONCLUSION
A novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

Keyword

Monoclonal antibody; CD34; Class III epitope; 27H2; Leukemia; Diagnosis

MeSH Terms

Animals
Antibodies, Monoclonal
Bone Marrow
Cell Line
Humans
Leukemia
Metalloendopeptidases
Mice
Neuraminidase
Palatine Tonsil
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Venules
Antibodies, Monoclonal
Metalloendopeptidases
Neuraminidase

Figure

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  Figure 1 Immunoreactivity of 27H2 on frozen tonsil. Frozen tonsil sections were immunohistochemically stained with monoclonal antibody 27H2 and counter-stained with hematoxylin. 27H2mAb show strong immunoreactivity on endothelial cells of high endothelial venules (Arrow, A), but mature lymphocytes (A) and squamous epithelia (Arrow head, B) reveal negative immunoreactivity (A: ×200, B: ×40).

• 

  Figure 2 Conformation of antigen recognized by 27H2 mAb as CD34. For searching of 27H2 mAb recognized antigen, TF-1 cells (lane 1, 2) and Bone marrow cells of acute myeloid leukemia patient (lane 3, 4) were lysed. Lysates were incubated with 27H2 mAb (lane 1, 3) and isotype matched control anti-CD120a (2, 4) coated protein G sepharose bead. The immunoprecipitates were separated by SDS-PAGE and electro-blotted with 5% skim milk. Membrane was incubated with anti-CD34 mAb (4H11), followed by peroxidase-conjugated goat anti-mouse Ig. Immunoreactive proteins were visualized using the enhanced chemiluminescence detection system. Note discreate band of about 100 kDa sized as expected in lane 1 and 3 (Arrows).

• 

  Figure 3 Identification of class III epitopes of CD34 by neuraminidase and endopeptidase treatment in TF-1 cells. (A) Identification of epitope using neuraminidase enzyme treatment. 27H2 showed neuraminidase resistant class II or III epitope, while. K06 revealed neuraminidase sensitive epitope. (B) Identification of epitope using O-sialoglycoprotein endopeptidase enzyme treatment. 27H2 showed endopeptidase resistant class III epitope, while. QBEnd 10 revealed endopeptidase sensitive class II epitope.

• 

  Figure 4 Identification of class III epitopes of CD34 by blocking assay with class III anti-HPCA-2 in TF-1 cells. Blocking assay with anti-HPCA-2 and 27H2. TF-1 cells were preincubated with various amounts of anti-HPCA-2 and then FITC conjugated 27H2 was incubated as detection antibody. Note competitive inhibition of class III anti-HPCA was definitive. The data are representative of three separate experiments performed.

• 

  Figure 5 Comparison with FITC conjugated commercial class III CD34 mAb (Anti-HPCA-2) in bone marrow cells of pre B cell acute lymphoblastic leukemia patient. Shown are the determinations of leukemic cells from a patient with pre B cell acute lymphoblastic leukemia. Staining was done with IgG FITC, PE, 27H2-FITC, Anti-HPCA-2-FITC, 4H11-FITC and HLA-DR (YG18)-PE. Note similar sensitivity for detecting leukemic cells of pre-B cell acute lymphoblastic leukemia patient.

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