Hiv 1 p24 elisa kit

The NLFOA procedure was similar to that in the classical HIV-1 p24 ELISA (PerkinElmer, Waltham, MA, U.S.A.), except that Streptavidin (SA)-HRP and OPD (o-phenylenediamine dihydrochloride) were replaced with nanoparticle-streptavidin (Innova Biosciences, Cambridge, Cambs, U.K.), biotinylated TurboNuclease (bio-TurboNuclease) and the FLOS substrate (Fig 1). Bio-TurboNuclease was prepared using the ImmunoProbe Biotinylation Kit (Sigma Aldrich, Saint Louis, MO, U.S.A.). The 96-cell polystyrene microplate was coated with the p24 monoclonal antibody (from PerkinElmer HIV-1 p24 ELISA kit) and blocked with blocking buffer (PBS containing 5.0 g/L defatted milk powder). After p24 was added and the plate was incubated at 37°C for two hours, the biotinylated anti-p24 antibody (from PerkinElmer HIV-1 p24 ELISA kit) was added and incubated at 37°C for one hour. Nanoparticle-streptavidin was then added and incubated at 37°C for 40 minutes. After bio-TurboNuclease was added and incubated at 37°C for 40 minutes, FLOS in TurboNuclease reaction buffer (50 mM Tris-HCl, pH 8.0 and 1 mM MgCl₂) was added and incubated at 37°C for 40 minutes. The fluorescence intensity was measured on a VICTOR X2 Series Multilabel Plate Reader (PerkinElmer, Waltham, MA, U.S.A.). A washing step with PBS containing 0.2% Tween 20 was performed between each step.