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Fig. 3 | Respiratory Research

Fig. 3

From: Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Fig. 3

The isolation of scFvs that bind to mast cells. a Binding of polyclonal phage-displayed antibodies to HMC-1. Three rounds of panning were carried out, with each round including a negative selection of phage against epitope-masked HMC-1 cells, followed by a positive selection against unmasked HMC-1. HMC-1 cells were detected with 1010 phage from panning rounds 0, 1, 2, and 3 (R0 to R3). Bound scFv was detected with biotin labelled anti-fd antibody and then extravadin-AP conjugate. Absorbances were measured at 405 nm. Individual scFvs that bound to HMC-1 cells were selected by phage ELISA. b and c scFv binding was confirmed by flow cytometry. HMC-1 or Jurkat cells were incubated with purified scFv and binding was visualised with anti-C-myc 9E10 antibody and anti-mouse antibody-FITC conjugate. The mean fluorescence produced with each scFv is expressed as a fold-increase from that produced in the absence of scFv b. ScFvs A1, A3, A8, B1, C1 and D1 were selected against HMC-1; the scFv E4 is a control scFv which does not bind to HMC-1. Representative examples of flow cytometry histograms for the binding of scFvs to HMC-1 and Jurkat cells are shown for antibody A1 and for a no scFv control c. The binding of scFvs to HMC-1 was repeated a minimum of 4 times and all 6 scFvs demonstrated clear binding to HMC-1 cells on each occasion. d scFv A1 also bound to HLMCs when compared to E4 (representative of 3 independent experiments using 3 HLMC donors)

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