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Microbes & Immunity A novel anti-EphA8 monoclonal antibody

3.3. Specificity of Ea Mab-9 to Eph receptor- 3.4. Determination of the binding affinity of
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expressed CHO-K1 cells Ea Mab-9 by flow cytometry
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CHO-K1 cells overexpressing all Eph receptors, including The binding affinity of Ea Mab-9 was assessed with
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EphA1 to A8, EphA10, EphB1 to B4, and EphB6, were exogenously EphA8-expressed CHO/EphA8 and
established. Using these fourteen cell lines, the specificity of LN229/EphA8 cells using flow cytometry. Results showed
Ea Mab-9 was evaluated. As shown in Figure 3, Ea Mab-9 that the K values of Ea Mab-9 were 1.3 × 10 M for
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D
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recognized CHO/EphA8 cells but did not bind to any CHO/EphA8 cells and 1.6 × 10 M for LN229/EphA8 cells
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other Eph receptor-expressed CHO-K1 cells. These results (Figure 4). The reproducibility of the binding data was
confirm the high specificity of Ea Mab-9 for EphA8 among confirmed (Figure A1). These results demonstrate that
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the Eph receptor family. Ea Mab-9 possesses a high affinity for EphA8.
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Figure 3. Flow cytometry of Ea Mab-9 in Eph receptor-expressed CHO-K1 cells. CHO-K1 cells transfected to express each of the fourteen Eph receptors
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were treated with 10 μg/mL of Ea Mab-9 (red line) or control blocking buffer (black line), followed by the treatment with Alexa Fluor 488-conjugated
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anti-mouse IgG.
Abbreviations: CHO: Chinese hamster ovary; EphA8: Erythropoietin-producing hepatocellular receptor A8; IgG: immunoglobulin g.
Volume 2 Issue 4 (2025) 155 doi: 10.36922/MI025060010