Enzyme-Linked Immunosorbent Assay for Plasminogen Binding

ELISA plate binding assay was used to determine the binding activity of rCpEno to human plasminogen as described by Bao et al. [16 (link)]. Briefly, 10 μg/well of purified rCpEno was added into 96-well plate and incubated at 4 °C overnight. BSA (10 μg/well) was used as a negative control. After being washed three times with PBST, the plate wells were blocked with 5% skim milk (Oxoid) in PBST at 37 °C for 2 h. After washing, different concentrations of human plasminogen (0, 1, 5, 10, 15, 20, 25 and 30 μg/ml in PBST) (Merck Millipore) were added to each well and incubated at 37 °C for 2 h. Then the plates were incubated with rabbit anti-plasminogen polyclonal antibody (100 μl/well, 1: 3,000) (Abcam, MA, USA) at 37 °C for 1.5 h. After washing three times with PBST, the plate wells were incubated with goat anti-rabbit IgG-HRP (100 μl/well, 1: 5,000) (Sigma-Aldrich) at 37 °C for 1 h. Finally, the colour reaction was performed by adding soluble TMB substrate solution and terminated with 2 M H₂SO₄. Absorbance at OD450 was read using a spectrophotometer (BioTek).