Alliance® HIV-1 p24 ELISA kit for detection and quantification of the major structural core component of HIV-1 virus, p24 antigen, was purchased from PerkinElmer Life Sciences (Boston, MA). Pathogen-free human serum and OPD substrate tablets were obtained from Sigma (St Louis, MO). Standard 96-well clear microplates were obtained from R&D Systems (Minneapolis, MN). A Labsystems Multiscan® MCC/340 microplate reader was used for optical density measurements.
Alliance hiv 1 p24 elisa kit
Manufactured by PerkinElmer
Sourced in United States
The Alliance HIV-1 p24 ELISA kit is a laboratory diagnostic tool designed for the detection of the HIV-1 p24 antigen. The kit utilizes an enzyme-linked immunosorbent assay (ELISA) technique to quantitatively measure the presence of the HIV-1 p24 antigen in biological samples.
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Lab products found in correlation
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96 well immunosorbent plates, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Taqman fast virus 1 step master mix, by Qiagen (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
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12 protocols using alliance hiv 1 p24 elisa kit
1
HIV-1 p24 ELISA Protocol
2
Quantification of Human IL-21 and HIV-1 p24
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Human IL-21 protein was determined in BLT mice plasma or culture supernatant using the human IL-21 ELISA MAX Deluxe Set (Biolegend; #433804). HIV-1 p24 antigen in HLAC culture supernatants was determined using the Alliance HIV-1 p24 ELISA kit (NEK050001; Perkin Elmer). Samples were assayed in duplicates and concentrations determined off of a standard curve.
3
Preparation and Quantification of HIV-1 YU2 Viral Stocks
4
Semi-Quantitative p24 ELISA for HIV-1 Fraction Assessment
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To define HIV-1 particle containing fractions and control purification efficiency, we performed a semi-quantitative p24 ELISA on the fractions using the Alliance HIV-1 p24 ELISA kit (Perkin Elmer). For this assay, we boiled 50 μl of each fraction at 95°C for 5 minutes prior to transfer to immunosorbent 96-well plates (NUNC, Denmark). After sealing the plates were incubated overnight at 4°C. Next, we removed the supernatant and blocked non-specific signal with 5% donkey serum for 30 minutes at room temperature. The subsequent experimental procedures and plate reading were performed following the manufacturer’s instructions.
5
Lentiviral Transduction and Safety Assurance
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Lentiviral vectors used have a self-inactivating (SIN) design and were produced in a BSL-2+ laboratory (49 (link)). Transduced cells were cultured in a BSL-2+ lab until the supernatant showed to be p24 ELISA negative (no residual LV or replication competent lentiviruses formed; Alliance HIV-1 p24 ELISA kit: Perkin Elmer), ~3 weeks after transduction, before being used for experiments. All experiments not requiring a confocal microscope were performed in a BSL-2+ lab.
6
ELISA-based Quantification of LVV Particles
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Quantification of the p24 capsid protein in LVV preparations was performed by an enzyme-linked immunosorbent assay (ELISA). p24 concentrations were measured with an Alliance HIV-1 p24 ELISA kit (Perkin Elmer, Waltham, MA, USA) and converted into total number of LVV particles using Eq. (2 ).
Molecular weight of p24: 2.4 × 104.
Number of p24 molecules per HIV particle: 2000.
Molecular weight of p24: 2.4 × 104.
Number of p24 molecules per HIV particle: 2000.
7
Lentiviral Challenge for Aptamer-shRNA Fusion
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For lentivirus challenge, 5 × 104 HEK293 cells stably expressing aptamer alone, shRNA alone or shRNA–aptamer fusion were transduced with HIV7-GFP (29 (link)) lentiviral particles at a MOI of 0.3 in 500 μl medium. Cells were harvested 10 days post-transduction and analyzed by FACS. For HIV challenge, 5 × 104 Ghost3 cells stably expressing shRNA alone or shRNA–aptamer fusions were infected with HIV-1 Ba-L strain at MOI of 0.02 in 500 μl medium. Viral concentration in culture was determined by p24 assay using Alliance HIV-1 p24 ELISA kit (PerkinElmer, Waltham, MA, USA).
For long term HIV challenge, 1 × 105 CCRF-CEM cells stably expressing shRNA or shRNA–aptamer fusions were infected with HIV-1 NL4-3 viruses at MOI of 0.02 in 500 μl medium. On Day 3, cells were collected by centrifugation at 200 × g for 3 min and resuspended in 1ml fresh medium. From week 1 to week 6 post-infection, 500 μl of cell cultures were collected for analysis and the continuing cultures was replenished with 500 μl fresh medium. In addition, to maintain an unsaturated cell density, 50% of cells was also replaced with fresh medium at mid-week time points.
For long term HIV challenge, 1 × 105 CCRF-CEM cells stably expressing shRNA or shRNA–aptamer fusions were infected with HIV-1 NL4-3 viruses at MOI of 0.02 in 500 μl medium. On Day 3, cells were collected by centrifugation at 200 × g for 3 min and resuspended in 1ml fresh medium. From week 1 to week 6 post-infection, 500 μl of cell cultures were collected for analysis and the continuing cultures was replenished with 500 μl fresh medium. In addition, to maintain an unsaturated cell density, 50% of cells was also replaced with fresh medium at mid-week time points.
8
HIV-1 Infection of HeLa P4 Cells
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HIV-1 infection of HeLa P4 cells was performed as described before [7 (link)]. A total of 3 × 104 cells per well were infected with HIV(NL4.3) in a6 well plate for 6 h. After 6 h, the virus was washed away with PBS (Invitrogen, Waltham, MA, USA) and replaced with 5 mL of medium. Replication of HIV-1 was monitored by quantifying p24 antigen in the supernatant via ELISA (Alliance HIV-1 p24 ELISA kit; PerkinElmer, Mechelen, Belgium).
9
HIV-1 Infection of Humanized Mice
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A cell-free virus stock of CCR5-tropic HIV-1 clone Bal was obtained from the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH. HIV-1 Bal virus was propagated in human peripheral blood mononuclear cells and harvested 10 days' post-infection. Virus was titrated using the Alliance HIV-1 p24 ELISA kit (PerkinElmer, Waltham, MA).
Hu-NSG mice with stable human leukocyte reconstitution (with a 20% or higher percentage of human CD45+ T cells) were infected with the HIV-1 Bal (200 ng p24/mouse) by intraperitoneal injection (i.p.) while under inhalant general anesthesia. Plasma viremia was assayed using one-step reverse transcriptase real-time PCR 22 (link) with automated CFX96 TouchTM Real Time PCR Detection System (Bio-Rad). HIV-1 levels in peripheral blood were determined by extracting RNA from 5×105 cells using the QIAamp Viral RNa mini kit (Qiagen) and performing Taqman qPCR using a primer and probe set targeting the HIV-1 LTR region, using the TaqMan Fast Virus 1-Step Master Mix as per the manufacturer's recommendations. The limit of detection in this assay is 40 copies/mL or less, but due to dilution, the limit of detection of our assays was 500-800 copies/mL, depending on the available sample volume.
Hu-NSG mice with stable human leukocyte reconstitution (with a 20% or higher percentage of human CD45+ T cells) were infected with the HIV-1 Bal (200 ng p24/mouse) by intraperitoneal injection (i.p.) while under inhalant general anesthesia. Plasma viremia was assayed using one-step reverse transcriptase real-time PCR 22 (link) with automated CFX96 TouchTM Real Time PCR Detection System (Bio-Rad). HIV-1 levels in peripheral blood were determined by extracting RNA from 5×105 cells using the QIAamp Viral RNa mini kit (Qiagen) and performing Taqman qPCR using a primer and probe set targeting the HIV-1 LTR region, using the TaqMan Fast Virus 1-Step Master Mix as per the manufacturer's recommendations. The limit of detection in this assay is 40 copies/mL or less, but due to dilution, the limit of detection of our assays was 500-800 copies/mL, depending on the available sample volume.
10
Quantification of Human IL-21 and HIV-1 p24
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