Target-binding activity of surrogate scFv isolated by Hishot display.

<p>Binding of surrogate scFv was measured by ELISA using horseradish peroxidase-conjugated anti-His-tag mAb. The ELISA plate was coated with proteins (5 µg/mL) and then blocked with BSA. Values represent the mean ± S.D. of triplicate determinations. (A) Specific binding activity of surrogate scFv against intelectin-1. The amino acid sequence of the random region is shown under the clone name (IA04 and IA10). Fixed Tyr is indicated as shaded characters. (B) Dose-dependency of the binding of purified surrogate scFv against intelectin-1. Values represent the mean of duplicate determinations. (C) Sandwich ELISA against intelectin-1, using surrogate scFv against intelectin-1 as the detector. Anti-intelectin-1 (capture mAb, 10:2D2) was coated at a concentration of 2 µg/mL, and intelectin-1 (0.1 µg/mL) was captured. The surrogate scFv against intelectin-1 was used at the concentration of 8 µg/mL. (D) Specific binding activity of surrogate scFv against TNF-α. The amino acid sequence of the random region is shown under the clone name (T4E). Fixed Tyr is indicated as shaded characters. BSA, bovine serum albumin; OVA, ovalbumin; Trf, transferrin; IgG, rabbit IgG; ITLN1, human intelectin-1; TNF-α, human tumor necrosis factor-α.