Figure 4. Purification and ELISA binding assay of isolated domain III specific binders D6 and D7. (A) SDS-PAGE analysis of D6 and D7 scFv-Fc fusion proteins. Lane 1-D6 scFv-Fc; Lane 2-D7 scFv-Fc. ELISA binding assessment of (B) D6 scFv-Fc and (C) D7 scFv-Fc to c-Myc tagged domain III.1–4-Fc proteins. D6 scFv-Fc and D7 scFv-Fc were each coated on plate at 2 μg/ml; DENV domain III.1–4-c-Myc tag was added as antigens and binding was detected via mouse anti-c-Myc-HRP. (D) Competition ELISA showing the binding epitopes of D6 scFv-Fc and D7 scFv-Fc overlap. D6 scFv-Fc was coated on plate, serially diluted c-Myc tagged domain III.3-Fc only or mixed with a constant amount of competitor (K310E mutant or D7 scFv-Fc) was added to each well and binding of the domain III.3-Fc was detected by mouse anti-c-Myc-HRP antibody.