Immune Netw.  2014 Jun;14(3):164-170. 10.4110/in.2014.14.3.164.

Characterization of Two Novel mAbs Recognizing Different Epitopes on CD43

• Kim S
• Hong JW
• Cho WD
• Moon YR
• Yoon SS
• Kim MY
• Hong KP
• Lee YM
• Yi JH
• Ham YJ
• Rah HC
• Kim SR
• Song HG

• KMID: 2168028
• DOI: http://doi.org/10.4110/in.2014.14.3.164

Abstract

JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.

Keyword

Leukemia; CD43; Epitope; Enzyme-Linked Immunosorbent Assay; Diagnosis

MeSH Terms

Antibodies, Monoclonal
Blotting, Western
Cell Line
Clone Cells
Diagnosis
Enzyme Assays
Enzyme-Linked Immunosorbent Assay
Epitopes*
Leukemia
Lymph Nodes
Thymus Gland
Antibodies, Monoclonal
Epitopes

Figure

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  Figure 1 Selection of YG5 pairing mAb to detect JL1 by sandwich ELISA. Sandwich ELISA was used to screen for clones, 2C8, 8E10, and 8B6, which resulted in a high OD (A). The final clones, 2C8-2-6 and 8E10-1-23, were selected in an experiment by limiting dilution (B). Both antibodies were suitable for use as a detector antibody in sandwich ELISA, when YG5 was used as the capture antibody.

• 

  Figure 2 Generation of recombinant CD43-hFC protein. To identify the antigen recognized by 2C8 and 8E10, CD43-hFC recombinant protein was generated. Schematic representation of gene cloning strategy (A). Protein levels confirmed by sandwich ELISA using anti-human Fc antibody as the capture and detector antibody (B). Purified CD43-hFC recombinant protein was detected by conventional ELISA, when YG5 was used as the detector antibody (C).

• 

  Figure 3 Identification of antigen recognized by 2C8 and 8E10 by western blot analysis. CD43-hFC recombinant protein was separated on 8% SDS-PAGE gel and detected using YG5, 2C8, 8E10, and DFT-1 mAbs at approximately 150 kDa under non-reducing conditions. CD43-hFC recombinant protein was detected at 50~60 kDa by all the anti-CD43 mAbs except 8E10 mAb under reducing conditions. NR: non reducing condition R: reducing condition.

• 

  Figure 4 Characterization of CD43 epitopes recognized by anti-CD43 mAbs. Treatment with neuraminidase reveals epitopes that are sensitive to the treatment. The epitope recognized by YG5 mAb was neuraminidase-independent, but the epitopes recognized by 2C8, 8E10, and DFT-1 mAbs were neuraminidase-sensitive.

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  Figure 5 The tissue distribution pattern of antigens recognized by anti-CD43 mAbs. YG5 showed strong immunoreactivity in the cortex of thymus, but lymph node remained unstained. 8E10 and DFT-1 displayed strong immunoreactivity on the mature T cell zone of the lymph node. In contrast, 2C8 displayed weak immunoreactivity in the cortex of thymus and the lymph node (×200). C: cortex, M: medulla, MTZ: mature T cell zone, F: follicle, GCT: germinal center of T cell.

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