Binding of aVn to recombinant Msf constructs.

A) ELISA plates were coated with Msf constructs (3 μM) and overlaid with aVn (74 nM). Bound aVn was detected using polyclonal rabbit anti-vitronectin antibody followed by alkaline phosphatase conjugated anti-rabbit antibody (grey columns). In control experiments, aVn was omitted (white columns). Each experiment was performed three times incorporating triplicate determinations within each experiment. Overall means (n = 3) and SD are shown. Msf_1–422, Msf_1–320, Msf_1–203, Msf_1–86 and Msf_39–124 were found to bind similar and significantly higher levels of aVn compared to Msf_197–422, Msf_pΔ4–82 or Msf_81–203. *P<0.05, ***P<0.0001 as indicated. B) ELISA plate coated with H44/76 Msf_1–86 and the equivalent region from Strain B16B6 and overlaid as A) above. No significant difference in binding between Msf from the two strains was observed (n = 3 ± SE are shown). C) Binding of recombinant Msf constructs to aVn. ELISA plates coated with aVn (74 nM) were overlaid with the Msf constructs (3 μM). Bound recombinant Msf was detected using rabbit anti-Msf polyclonal antiserum followed by alkaline phosphatase conjugated anti-rabbit antibody. Msf_1–422, Msf_1–203 and Msf_1–320 bound at similar levels to immobilised aVn compared with Msf_197–422 which was approximately 6 fold lower compared with Msf_1–422. The Δ4–82 derivative of Msf_1–422 also lost aVn binding capacity which was reduced by ~4-fold. Mean values of three independent experiments ± SD are shown. *P<0.05. D) Control experiment to establish equivalent detection of Msf constructs using anti-Msf polyclonal antibody. Recombinant Msf constructs coated onto the ELISA plate (3μM) were overlaid with anti-Msf polyclonal antibody and anti-rabbit alkaline phosphatase conjugated secondary antibody. Plates were subsequently developed with pNPP substrate (Sigma). Data shown are means of triplicate determinations within a single experiment. E) ELISA wells were either coated with Msf_1–422 (3 μM) or uncoated prior to blocking. Wells were overlaid with polyclonal rabbit anti-vitronectin followed by alkaline phosphatase conjugated anti-rabbit antibody prior to developing. Means of three independent experiments ± SE are shown. F) ELISA plates were coated with recombinant Msf proteins (3μM), overlaid aVn (74nM) and bound aVn detected with anti-vitronectin polyclonal antibody and anti-rabbit alkaline phosphatase conjugated secondary antibody (grey columns). An unrelated protein (CEACAM1 N-Fc) was used in the experiment as a control to determine nonspecific aVn binding (white column). Data shown are means of triplicate determinations within a single experiment. G) Size exclusion chromatograms prepared using an S200 column for Msf_1–422, _1–203 and _1–86 as indicated. Peak elution volumes and predicted molecular weights were obtained as follows Msf_1–422 59.3 ml and 270 kDa, Msf_1–203 76.1 ml and 67 kDa and Msf_1–86 84.1 ml and 34.5 kDa indicative of oligomeric forms of each construct. H) Western blot of unheated Msf recombinant proteins (as indicated) following SDS-PAGE. Msf was detected in each instance using anti-Msf polyclonal antibody raised in rabbit followed by anti-rabbit-alkaline phosphatase conjugated antibody prior to chromogenic development. Notably oligomeric bands were detected in the region approximating a trimeric molecular weight for each protein (*). In panel A and B the black line indicates the level of aVn binding observed to the unrelated control protein (Fig 2F).