Fig. 4
Characterization of the purified 4E5 mAb. a The specificity of the 4E5 mAb was determined by indirect ELISA (BCL6: B-cell lymphoma 6 protein, PD1-L1: Programmed cell death 1 ligand 1, PDPN: Podoplanin, PGY: P-glycoprotein). b The specificity of the 4E5 mAb was determined by Western Blot. Lane M: Marker, Lane 1–2: His-CgA protein duplicates, Lane 3: Random control (His-PD1-L1 human protein), Lane 4–6: The result of Western Blot. The proteins in Lane 4 and 5 are his-CgA protein duplicates, and Lane 6 was random control (His-PD1-L1). c Affinity of 4E5 mAb was analyzed by iELISA. Different concentrations (12.5, 25, 50, and 100 ng/mL) of coating antigen (CgA-His) were used to determine the affinity constant which is 9.23 × 109 L/mol. d Affinity of control antibodies (LK2H10 and PHE5) was analyzed by iELISA. Different concentrations (1, 1.5, and 2.5 μg/mL) of coating antigen (CgA-His) were used to determine the affinity constant which is 3.89 × 108 L/mol. e Epitope of 4E5 mAb was determined with western blot. Lane1–7: the result of SDS-PAGE analysis. Lane M: Marker, lane 1: the total protein of BL21 (DE3) with empty vector pET-28a, lane 2: the total protein of BL21 (DE3) with pET-28a-CgA fragment 1 (F1), lane 3: CgA fragment 2 (F2), lane 4: CgA fragment 3 (F3), lane 5: CgA fragment 4 (F4), lane 6: CgA. Lane 7–12: The result of Western Blot. Lane 7: the total protein of BL21 (DE3) with empty vector pET-28a, lane 8: CgA fragment 1 (F1), lane 9: CgA fragment 2 (F2), lane 10: CgA fragment 3 (F3), lane 11: CgA fragment 4 (F4), lane 12: CgA