Neutralite avidin peroxidase

High binding ELISA plates (ThermoFisher, Waltham, MA) were coated at 1μg/mL with goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL) overnight at 4°C. The next day, plates were washed 6x with 0.05% PBS-tween (PBST) and blocked with 1% BSA in PBST for 30 minutes at RT. Mouse anti-p27 2F12 antibody (AIDS reagent Program), at 1μg/mL, was added to plates and incubated for 1 hr at 37°C. After washing plates 6x, p27 standard (Immune Technologies) and culture medium treated with 0.5% Triton X-100 detergent (Sigma) to lyse virus particles were added in 3-fold dilutions and allowed to incubate for 1.5 hr at 37°C. Plates were washed and biotinylated SIV IG diluted 1:1000 (prepared in the laboratory) and added to each well. After 1 hr at 37°C, plates were washed, and 1:4000 neutralite-avidin peroxidase (Southern Biotech) was added. After 30 min at RT in the dark, bound IgG was detected using tetramethylbenzidine substrate (KPL, Gaithersburg, MD). The reaction was stopped by adding 100 μl of 2N H₂SO₄.

Concentrations of p24 were measured using a p24 ELISA, as previously described [48 (link)]. Briefly, high protein binding microtiter plates (ThermoFisher) were coated overnight with an anti-p24 monoclonal antibody that had been purified from H12 hybridoma (ARP) culture medium using Protein G Sepharose (ThermoFisher). Plates were washed with PBS containing 0.05% Tween-20 (PBST) and blocked with PBST containing 5% nonfat dry milk and 1% FBS. Samples were serially diluted in 1% FBS/PBST and added to plates. A recombinant HIV p24 protein (ImmunoDiagnostics, Woburn, MA) previously calibrated using the PerkinElmer HIV p24 ELISA kit was used to generate a standard curve. Following an overnight reaction at 4°C, the plates were washed and treated for 1 h at 37°C with HIVIG (ARP) that had been biotinylated in the lab using EZ Link Sulfo NHS (ThermoFisher). The plates were then washed and consecutively treated for 30 min at room temperature with neutralite avidin-peroxidase and tetramethylbenzene (both SouthernBiotech, Birmingham, AL). After addition of H₂SO₄ stop solution, absorbance was recorded at 450nm. The concentration of p24 in samples was then interpolated from standard curves constructed with the SoftMax Pro computer program (Molecular Devices, Sunnyvale, CA).