Hiv 1 p24 antigen elisa

DCs or Møs (1x10⁶ cells/mL) were exposed to the following protease inhibitors for 45 minutes: 10μM MG132, 5μM E64, 10μM cathepsin S inhibitor Z-FL-COCHO, 10μM leupeptin (Enzo Life Sciences), 120μM bestatin (Sigma-Aldrich), before incubation with different concentrations of recombinant HIV-1 p24-protein for 1hr at 37°C or 4°C. Samples were thoroughly washed in ice-cold PBS and immediately treated with 3mg/mL pronase E (Sigma-Aldrich) in AIM-V media without serum for 10 minutes on ice. Cells were washed, lysed in 0.5% Triton X-100 containing lysis buffer and the amount of p24 protein in cell lysates was measured using a standard HIV-1 p24 antigen ELISA (Perkin Elmer).

HIV-infected peripheral blood monoclonal cells (PBMCs) were first isolated from patient blood samples using Ficoll-Hypaque density gradient cell centrifugation. PBMCs were then stimulated by phytohemagglutinin and co-cultured with irradiated PBMCs at 37 °C and 5% of CO₂. The virus titer in the co-culture supernatant was tested using HIV-1 p24 antigen ELISA (PerkinElmer Life Science, Inc., NEK050b). The co-culture process was continued till the concentration of p24 became 20 ng/ml. The cell culture supernatant was collected, and the virus concentration was tested using Roche-COBAS AmpliPrep TaqMan HIV-1 v2.0 system at Brigham and Women’s Hospital (BWH). For sample testing with the cellphone system, HIV-1 RNA was isolated from each sample using the AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) following the manufacturer's protocol.