Fig. 2

Polyclonal phage ELISA, monoclonal phage ELISA, and VHH purification and characterization. a: Polyclonal phage ELISA assessment of the outputs of each round of the biopanning process. The values are the mean of at least three replicates (p < 0.05). b: Monoclonal phage ELISA of the 4th round of biopanning. 60 random clones were selected and their binding capacity to CD19 was assessed using ELISA. The clones which exhibited a higher value of absorbance ratio to the control group (BSA) were selected for other in-depth characterization steps (GR37 and GR41). The values are presented as the mean of at least three replicates. c: SDS-PAGE analysis of purified GR41 expressed by pET-26b(+)-GR41-harboring BL21 (DE3) bacterial cells. Lane 1: IPTG-induced (1 mM) bacterial lysate, Lane 2: The supernatant from the culture media of IPTG-induced (1 mM) bacterial cells, Lane 3: The supernatant from the sonicated IPTG-induced (1 mM) bacterial cells, Lane 4: The flow-through fraction of IPTG-induced bacterial cell supernatant after being loaded onto a Ni–NTA chromatography column and passing through it, Lane M: protein marker, Lane 5: Column washing with 50 mM imidazole, Lane 6 and 7: Elution with 300 mM imidazole (circled red), Lane 8: Elution with 400 mM imidazole (circled red), Lane 9 and 10: Elution with 500 mM imidazole (circled red). d: Affinity determination of GR37 and GR41. The K_aff of GR37 was determined as 1.15 × 10⁷ M⁻¹ whereas the K_aff of GR41 was calculated as 2.08 × 10⁷ M⁻¹. e: Binding specificity of GR37 and GR41 to CD19 as determined by ELISA compared to the irrelevant proteins HER2, MUC1, BSA, and Ovalbumin. The values are the mean of at least three replicates (p < 0.05). f: The sensitivity profile of GR37 and GR41 to CD19. The results indicated that both VHHs are capable of detecting CD19 in a concentration as low as 2 ng/mL. The data are the mean of three replicates. * for p values < 0.05, **** for p values < 0.0001, and ns for p values > 0.05