Quantitative Immunoassay for Viral Antibodies

At the end of the incubation period, the plates were rinsed three times with Washing Buffer and then treated with 100 μl of 2,2′-azino-bis 3-ethylbenzthiazoline- 6-sulfonic acid (ABTS) (Sigma-Aldrich, Milan) in buffer solution (9.1 mM ABTS; pH 5.0), which reacted with the peroxidase enzyme to yield the color reaction. The plate was then read spectrophotometrically (Thermo Electron Corp., model Multiskan EX, Finland) at a wavelength (λ) of 405 nm. This optical density (OD) reading reflected the extent of immunocomplexes formed by the presence of specific antibodies, which bound to the SV40 synthetic peptide/epitopes/mimotopes. The three SV40 negative control sera were selected from samples below the cut-off value determined with second–degree polynomial regression by plotting the ranked net OD individual values for each peptide. A tendency curve was drawn from a second–degree polynomial regression for Tag A peptide and Tag D peptide, as published for MCPyV and BKPyV virus-like particles (VLPs) (Touzé et al., 2010 (link)). Our OD data/analysis revealed an inflection point corresponding to 0.18 for each peptide (Tognon et al., 2016 (link)).