Journal: Biochemistry and Biophysics Reports
Article Title: Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases
Figure Lengend Snippet: Immunological activity of the antibody preparations directed against human α-enolase (•―•) and β-enolase (о―о). ELISA plate wells were coated with 0.5 μg of purified α-, or β-enolase. The plates were washed, blocked with TBST and allowed to react with series of diluted, affinity chromatography purified, anti-human-α- and β-enolase antibody preparations. Goat anti-rabbit-IgG-HRP conjugate was used as the secondary antibody at 1:3000 dilution. Formation of the immunological complexes was tested by measuring HRP activity with o -phenylenediamine (OPD) as the HRP substrate. The progress of the HRP activity was monitored at 450 nm in a PerkinElmer microplate reader. Results were plotted and fitted into the linear regression equation. Slopes were calculated and compared. The data points are average of three experiments (n = 3) and average deviation from the mean for these data points was calculated at ± 0.015.
Article Snippet: Unoccupied protein binding sites on the polystyrene plate were blocked with 3% fat-free skimmed milk in TBS (20 mM Tris, 150 mM NaCl, pH 7.4) at 37 °C for 1 h. After washing with TBST, antibodies against human α-enolase or β-enolase were added in series of dilutions ranging from 0.003 μg/ml to 25 μg/ml and incubated at 37 °C for 2 h. The primary antibody was removed and after washing with TBST, the secondary antibody, goat anti-rabbit IgG-Horse Radish Peroxidase (HRP) conjugate (Promega) at 1:3000 dilution was added and incubated for 2 h. at 37 °C.
Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Purification, Affinity Chromatography