Fig 2: (A) Indirect enzyme-linked immunosorbent assay (ELISA) for determining the binding of HuscFvs expressed from various phage-infected E. coli clones to purified kaouthiagin. HuscFvs in lysates of E. coli clones no. 15, 20, and 61 gave significant ELISA signals (OD405nm) to kaouthiagin more than two times higher than bovine serum albumin (BSA) (antigen control). E. coli HB2151 (HB), lysate of normal E. coli HB2151 (background binding control). (B) Western blot patterns of kaouthiagin (~50 kDa) probed with lysates of E. coli clones 15, 20, and 61 which contained HuscFvs. The HuscFvs bound to the kaouthiagin and appear as bands at ~50 kDa. M, standard molecular weight proteins; +ve, kaouthiagin blotted strip probed with vWF (positive binding control); HB, kaouthiagin blotted strip probed with lysate of normal HB2151 E. coli (background control); Irr, kaouthiagin blotted strip probed with lysate of HB2151 E. coli containing irrelevant HuscFv (negative binding control).

Fig 3: (A) Sephacryl S-200 column chromatographic fractions no. 44–56 were separated by 14% under non-reducing condition and the presumptive kaouthiagin bands were revealed in fractions 46–52 at ~50 kDa after Coomassie Brilliant Blue G-250 (CBB) staining. (B) Western blotting patterns of the SDS-PAGE-separated fractions 44–56 probed with 1 μg/mL of recombinant human von Willebrand factor (vWF); the vWF bound to the separated venom components in fractions 46–52, indicating that vWF-bound protein is N. kaouthia kaouthiagin which was subsequently verified by LC-MS/MS and orthologous protein database search. (C) SDS-PAGE-separated-concentrated kaouthiagin after CBB staining (left panel) and Western blot pattern of the concentrated kaouthiagin probed with vWF (right panel). K, purified kaouthiagin with an apparent molecular weight of ~50 kDa (arrow). M of all panels, standard molecular weight proteins.