PMC

Outline of procedures of yeast two-hybrid human scFv library construction, library screening, and affinity maturation.

Alignment of amino acid sequences of V_H and V_L of the scFvs. A: Alignment of amino acid sequences of V_H and V_L of anti-IL8 scFv primary clones 4-123-36, 4-123-157, and 4-123-151. B: Comparison of primary scFv clone 4-123-36 and its affinity-maturated clones (M36-8 and M36-11). Residues identical to those of original clone 4-123-36 are shown by dashes. Dots represent gaps. Kabat CDR sequences are boxed.

ONPG assay of affinity-maturated clones. Plasmids from anti-IL8 scFv clone 4-123-36 and its affinity-matured clones (M36-8 and M36-11) were cotransformed into yeast with pGBK–IL8. The β-galactosidase activities were measured by quantitative ONPG assay. Activity is displayed as the fold of increase relative to the average of the parental clone 4-123-26. Error bars represent the standard errors from three experiments.

Coimmunoprecipitation of full human IgG antibody anti-human IL-8 by western blot. V_H and V_L regions of scFv clone 4-123-36 were cloned to the N-termini of human Ig γ1 heavy chain constant region and λ constant regions, respectively. The full human antibody constructs were transfected into COS cells. Full human IgG antibody was precipitated from the medium using beads coated with human IL-8 (lane 1) or uncoated beads (lane 2). Similarly, the medium of COS cells transfected with mock vectors was precipitated with IL-8-coated beads (lane 3). The blot was detected using HRP-conjugated goat anti-human IgG antibody.

ELISA analysis of primary and affinity-maturated scFvs against human IL-8. Human IL8 was coated on 96-well plates. Serially diluted scFv proteins were incubated in the IL8-coated wells in quadruplicates of each experiment. Binding was detected by HSV tag monoclonal antibody followed by anti-mouse IgG-HRP conjugate. The interaction was detected with TMB liquid substrates. Average readings of OD₄₅₀ in the figure are after subtraction of the background. Solid diamond (♦): the primary clone (4-123-36); solid square (▪): affinity maturated clone M36-8; solid triangle (▴): affinity maturated clone M36-11. Each point is the average of four experiments.

Coimmunoprecipitation of human IL-8 and its antibodies. ScFv protein was expressed in the periplasmic space of E. coli as a fusion with HSV and 6xHis tags at its carboxy terminus. They were used for coimmunoprecipitation experiments. Panel A: lane 1, total protein from periplasmic preparation; lane 2, scFv purified by Ni-NTA. Panel B: lane 1, western blot of total periplasmic preparation using antibody against HSV tag. Panel C: Reactigels were coated with hIL8 (lane 1), lamin C (lane 2), mouse p53 (lane 3), or no coating (lane 4) and then mixed with scFv anti-hIL8 (clone 123-36). After washing, the bound proteins were analyzed by western blot using antibody against HSV tag. Panel D: Reactigels were coated with scFvs of clones 4-123-36 (lane 1), M36-8 (lane 2), M36-11 (lane 3) or no coating (lane 4) and then mixed with hIL8 in the presence of BSA. After washing, the bound proteins were analyzed by western blot using mouse monoclonal antibody against hIL8. Open arrowhead points the scFv bands. Solid arrowhead points the hIL8 band, whereas the solid arrow points the band that is probably the dimer of the hIL8.

Inhibition of IL8-induced neutrophil chemotaxis by anti-IL8 antibodies. A: Purified scFv proteins were mixed with human IL8 and loaded at the bottom wells of Boyden chambers. Isolated neutrophil suspensions were added to upper chamber in each well. The chemotaxis was measured by counting neutrophil cells that migrated from the upper chamber to the low chamber. Percentages of inhibition are calculated based on the relative IL8-induced chemotaxis in the absence of scFv antibodies. Solid diamond (♦, fourth curve from the top): the primary clone (4-123-36); solid square (▪, second curve from the top): M36-8; solid triangle (▴, third curve from the top): M36-11; open triangle (▵, top curve): mouse anti-hIL8 monoclonal antibody (positive control); and cross (×, bottom curve): scFv anti-lamin (negative control). B: The V_H and V_L regions of primary clone were converted to full human IgG antibody (full Ab). The full Ab was transiently expressed in COS cells. Similarly to A, the culture medium (after fourfold concentration) was assayed for the inhibition of IL8-induced neutrophil chemotaxis. The positive control is mouse anti-hIL8 monoclonal antibody (mouse mAb) at 10 μg/mL, and the negative control is the culture medium of COS cells transfected with the empty vectors.