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.
Hiv 1 p24 elisa
Manufactured by PerkinElmer
Sourced in United States
The HIV-1 p24 ELISA is a laboratory equipment product designed to detect the presence of the HIV-1 p24 antigen in human serum or plasma samples. The product utilizes the enzyme-linked immunosorbent assay (ELISA) technique to quantitatively measure the HIV-1 p24 antigen levels.
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Lab products found in correlation
Foetal calf serum, by Biosera (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Hiv 1 p24 elisa kit, by PerkinElmer (1 mentions)
Immunoprobe biotinylation kit, by Merck Group (1 mentions)
P24 elisa assay, by PerkinElmer (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Ampkα2, by Merck Group (1 mentions)
Pha m, by Roche (1 mentions)
Hil 2, by Roche (1 mentions)
Dynabeads cd8, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
12 protocols using hiv 1 p24 elisa
1
Nanoparticle-Enhanced ELISA for p24 Detection
2
HIV-1 p24 Protein Quantification
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p24 was determined by PerkinElmer HIV-1 p24 ELISA following the manufacturer’s instructions.
3
HIV Virus Production and Quantification
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NL4-3 and NFNXS were prepared by calcium phosphate transfection of 293T cells with either NL4-3 or NFNXS plasmid DNA. VCM was collected 72 hours posttransfection, and aliquots stored at −80 °C.
BaL, SF162, Bru, 92UG021, and 92US723 were prepared by infecting cell-free supernatant from PBMCs onto fresh uninfected PBMCs in the presence of 8 µg/ml Polybrene. Infections were performed over 2–2.5 hours with gentle rocking, followed by one wash of the cells and culture in RPMI containing 20% fetal bovine serum and 100 U/ml IL-2. Fresh culture media and PBMCs were added from days 7 through 21. Supernatant was collected every 3 days from day 14, and aliquots stored at −80 °C.
BaL and SF2 used in Molt4/CCR5 experiments were prepared by infecting PM-1 cells with cell culture supernatant containing either BaL or SF2 in the presence of 8 µg/ml Polybrene. Infections were performed over 2–2.5 hours with gentle rocking. At completion of infection, cells were washed once and put into culture. Fresh media and PM-1 cells were added from days 7 through 21. Supernatant was collected every 3 days from day 14, and aliquots stored at −80 °C. Titers for all virus batches were determined using Perkin Elmer HIV-1 P24 ELISA.
BaL, SF162, Bru, 92UG021, and 92US723 were prepared by infecting cell-free supernatant from PBMCs onto fresh uninfected PBMCs in the presence of 8 µg/ml Polybrene. Infections were performed over 2–2.5 hours with gentle rocking, followed by one wash of the cells and culture in RPMI containing 20% fetal bovine serum and 100 U/ml IL-2. Fresh culture media and PBMCs were added from days 7 through 21. Supernatant was collected every 3 days from day 14, and aliquots stored at −80 °C.
BaL and SF2 used in Molt4/CCR5 experiments were prepared by infecting PM-1 cells with cell culture supernatant containing either BaL or SF2 in the presence of 8 µg/ml Polybrene. Infections were performed over 2–2.5 hours with gentle rocking. At completion of infection, cells were washed once and put into culture. Fresh media and PM-1 cells were added from days 7 through 21. Supernatant was collected every 3 days from day 14, and aliquots stored at −80 °C. Titers for all virus batches were determined using Perkin Elmer HIV-1 P24 ELISA.
4
Generating and Titrating Retroviral Vectors
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293T and TE671 cells were maintained in DMEM (Invitrogen) and U937 cells in RPMI +[L]-Glutamine (GIBCO), each supplemented with 10% heat inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma). U937 cells stably expressing SAMHD1 variants were prepared by transduction with MoMLV-based Puromycin-expressing VLPs, followed by selection with puromycin at 10 μg/mL. MoMLV-based YFP-expressing VLPs were made by cotransfecting 293T cells with pVSV-G (gifted from D. Lindemann), pKB4 [37 (link)] and pLGateway_SAMHD1IRESYFP (wild-type or mutants), harvesting 48 hr post-transfection. HIV-1GFP was produced by cotransfection of pVSV-G, p8.91 and pCSGW [38 (link)]. MoMLV-based Puromycin-expressing VLPs were made by cotransfection of pKB4, pVSV-G and pCMS28_SAMHD1 (wild-type or mutants). VLPs were titred on TE671 or 293T cells for normalisation prior to infection. Viruses with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA (Perkin Elmer).
5
HIV-1 Infection Assay in 293 T Cells
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293 T cells (2 × 106) were transfected with 2 μg of p125 (M-tropic, ADA strain) plasmids using Lipofectamine 2000, and the supernatants were harvested at 48 h post-transfection. Viruses were pretreated with 2 U/mL DNase I (Life Technologies) at 37 °C for 30 min before infection, and the viral titer was quantified using the p24 ELISA assay (PerkinElmer, Waltham, MA, USA). Cells with over-expressed or reduced levels of MRJ-L were titrated to adjust for equal cell numbers, and 1 × 106 cells were infected with M-tropic strain of HIV-1 (equivalent to 25 ng of p24 antigen) at 37 °C for 2 h. After washing three times with PBS, cells were maintained in RPMI1640/2% FBS and half of the medium was replaced every three days. Culture supernatants were collected every three days for quantification of p24 antigen by HIV-1 p24 ELISA (PerkinElmer).
6
Generating Viral-Like Particles for Cell Transduction
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293 T cells were maintained in DMEM (Invitrogen) and U937 and Jurkat T-cells were cultured in RPMI containing [L]-Glutamine (ThermoFisher) at 37 °C in 5% CO2. Media were supplemented with 10% heat-inactivated foetal calf serum (Biosera) and 1% penicillin/streptomycin (Sigma). MLV-based VLPs used to transduce cells with SAMHD1 were produced by co-transfecting 293 T cells with pVSV-G, pKB4 and pLGateway_SAMHD1IRESYFP (either WT or SAMHD1 mutant). HIV-1-GFP VLPs were produced by co-transfecting 293 T cells with pCSGW, pVSV-G, and p8.91 (either WT or RT mutant). SIVmac VLPs containing or lacking Vpx were produced by co-transfecting 293 T cells with pVSV-G and either pSIV3+ (for Vpx +) or pSIV3+vpx- (for Vpx-). VLP-containing cell culture supernatants were harvested 48 h post-transfection and titred on TE671 or 293 T cells for normalisation prior to infection. VLPs with mutations in Reverse Transcriptase were normalised by HIV-1 p24 ELISA (Perkin Elmer).
7
Lentiviral-mediated Knockdown of HDAC1/2
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Lentiviral-mediated shRNA knockdown was performed as described previously32 (link) using the MISSION shRNA methodology (Sigma). Virus titers were determined using the HIV-1 p24 ELISA (NEK050B001KT, Perkin-Elmer), according to the manufacturer’s instructions. The following pLKO.1 lentiviral vectors were used: TRCN0000039402 (shHDAC1), TRCN0000039397 (shHDAC2), shc002 (scrambled shRNA). Med1-MB cells and primary medulloblastoma cells were transduced with lentiviruses (MOI = 5), and incubated for the indicated times. Infected cells were selected with 5 μg/ml puromycin, where indicated.
8
Avirulin Inhibits HIV-1 Infection in PM1 Cells
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PM1 cells (3 × 106/mL) were incubated with Avirulin or equivalent vehicle and HIV-1 in RPMI with 2% serum (‘R2’) for 90 min. at 37 °C, 5% CO2 at a volume of 100 µL, then diluted with fresh R2 and spun at 200× g for 5 min. Cells were then resuspended in 500 µL fresh R20 with equivalent concentration of treatment. Infection with clinical isolates required 2 µg/mL polybrene, a cationic polymer that increase efficiency of retroviral infection. On days 3 and 6 post infection, supernatants were collected, live and dead cell number was determined using trypan blue staining. On day 3 post infection, cells were resuspended in fresh media and diluted to the initial cell concentration and retreated with drug or vehicle. Viral inhibition was determined by measuring concentration of p24 in cell supernatant per million live cells. p24 was detected by HIV-1 p24 ELISA (Perkin Elmer, Waltham, MA, USA).
9
Lentiviral Knockdown of AMPK Subunits
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Subconfluent HEK293T cells were cotransfected by calcium phosphate precipitation with 20 μg of PLKO.1, 15 μg pCMV-R 8.74 and 10 μg pMD.G, to produce shAMPKα1 or shAmpkα2 or non-targeting lentiviruses. Supernatant was collected 24 and 48 hours later. Virus titer was determined using the HIV-1 p24 ELISA (NEK050B001KT, Perkin-Elmer), following manufacturer's instructions. pLKO.1 vectors used for lentiviral production were obtained by Sigma (MISSION shRNA SIGMA): Ampkα1 (TRC0000000861), Ampkα2 (TRC00000002171) and shc002 (control shRNA). To perform the lentiviral transduction, DAOY cells were seeded overnight in 12-well plates. The day after, 5 MOI of lentivirus were complexed with 5 mg/ml polybrene (Santa Cruz Biotechnology), added to the cells and left 24 hours before being removed and replaced with standard medium. Knockdown efficiency was monitored by AMPK western blotting.
10
HIV Infection Kinetics in Stimulated PBMCs
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Isolated PBMCs were stimulated with 3 μg ml−1 of PHA-M (Roche) and 20 U ml−1 of human-IL 2 (hIL-2; Roche) for 3 days; viral supernatant from transfection of 293T cells was used to infect pooled mixed stimulated PBMCs prepared from two healthy donors. The supernatant from HIV-infected PBMCs was collected at day 7 and day 11. Viral titers were determined by HIV-1 p24 ELISA (PerkinElmer).
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