p24 ELISA

COSTAR white opaque 96-well ELISA plates (Corning) were coated overnight with affinity purified sheep anti-HIV-1 p24 gag antibody (Aalto Bio Reagents), washed three times with tris-buffered saline (TBS) and allowed to dry, as previously described (52). Plates were stored at −20°C for medium term use. A p24 standard dilution series was prepared in binding buffer made up of TBS with 1% Empigen (Sigma) after reconstituting bacterially expressed p24 (Aalto Bio Reagents) in TBS with 1% FCS. Virus containing sample supernatants were also serially diluted in binding buffer and 100 μL of samples and p24 standards were placed in pre-coated and blocked plates. Plates were covered and incubated for 2 h at room temperature. The plates were then washed three times with TBS. One hundred microliters of a 1 in 12,500 dilution of an alkaline phosphatase conjugated sheep anti-HIV-1 monoclonal antibody BC 1071-AP (Aalto Biosciences) in TBS with 0.1% TWEEN-20 was added and incubated for 1 h at room temperature. Wells were washed four times with a TBS buffer with TROPIX and 0.1% TWEEN. Bound antibodies were measured by placing 100 μL of a solution of CDP Star with Saphire-II (Life Tech) in TROPIX buffer in each well for half an hour and reading in a luminometer. All reactions were run in triplicate and the equation generated from the linear range of the p24 standard curve was used to determine the amount of p24 in each sample based on the results of the luminometer readings.