The levels of CCL2 present in culture supernatants were measured by ELISA kits purchased from R&D Systems (detection limit 5 pg/ml). Viral production was monitored by measuring HIV-1 p24 Gag Ag release by the INNOTEST HIV Antigen mAb kit (Innogenetics).
Innotest hiv antigen mab kit
Manufactured by Fujirebio
Sourced in Belgium
The INNOTEST HIV Antigen mAb kit is a diagnostic laboratory equipment product designed for the detection of the human immunodeficiency virus (HIV) antigen. It utilizes monoclonal antibodies (mAb) as the core detection mechanism.
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6 protocols using innotest hiv antigen mab kit
1
Measuring CCL2 and HIV-1 p24 Levels
2
Quantification of HIV-1 Production
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HIV‐1 production was measured in supernatants of the J‐Lat 8.4 and J‐Lat 15.4 cell cultures by the INNOTEST® HIV Antigen mAb kit according to the manufacturer's instructions (Innogenetics). INNOTEST® HIV Antigen mAb is an enzyme immunoassay for the detection of HIV p24 antigen in human serum, plasma, and cell culture supernatant.
3
Single-Round Infection Assay for HIV-1
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Infectious viral stocks were generated by transfecting the viral DNA into HEK293T cells and collecting supernatants after 48 h. Viral production was quantified in the supernatants for HIV-1 p24 antigen content using the Innotest HIV antigen mAB kit (Innogenetics N.V. Gent, Belgium). Before infection, the viral stock was treated with 40 U/ml DNAse I (Life Sciences) for 1 h at 25 °C. Activated T cells (1 × 106) were then infected with 500 ng/ml of p24 for 4–5 h at 37 °C. Infection was carried out for 4 h in the presence of polybrene (Sigma). After infection, the cells were kept in culture at 1 × 106 cells/ml in complete medium supplemented with IL-2. At days 3, 5, 7, 10 and 14 post-infection, media and IL-2 were replaced and cells were kept at a density of 1 × 106/ml. The Env- molecular clone pNL4–3/Luc E− R−, a kind gift from Nathaniel Landau, harbors a frameshift mutation introduced near the 5′ end of env gene (Connor et al., 1995 (link)), and performs a single-round infection once pseudotyped with the Vesicular Stomatitis Virus–G (VSV-G) protein. Integrated viral DNA was quantified by the Alu-PCR technique using a described procedure (Manganaro et al., 2010 (link)).
4
Pseudotyped HIV-1 infectivity assay
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20,000 HeLaP4 cells were seeded into 96-well plates on the day prior to infection. Cells were infected in triplicate with 3 dilutions (typically 1 × 105, 3.3 × 104 and 1.1 × 104 pg p24) of VSV-G pseudotyped single-round HIV-1 supplemented with one of the compounds in a total volume of 200 µl per well. The virus was produced as described previously [31 (link)]. The final compound concentration in the assay was 100 µM. p24 measurements were performed with the Innotest HIV Antigen mAb kit (Fujirebio). 24 h after infection the supernatant was replaced by fresh medium. 72 h post infection cells were lysed in 50 µl of lysis buffer (50 mM Tris/HCl, pH 7.3, 200 mM NaCl, 0.2% NP40, 5% glycerol) and analyzed for firefly luciferase activity (ONE-Glo™, Promega, Belgium) according to the manufacturer’s protocol. Chemiluminescence was measured with a Glomax luminometer (Promega, Belgium). The signals were normalized for protein content as determined by a BCA protein assay (Thermo Scientific Pierce).
5
HIV-1 p24 Gag ELISA Quantification
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HIV-1 production was measured in cell culture supernatants by ELISA assays on p24Gag using the INNOTEST HIV Antigen mAb kit according to the manufacturer's instructions (Fujirebio).
6
Western Blot and ELISA for HIV p24 Quantification
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HeLa cells were washed 24 h posttransfection with PBS and resuspended in ice-cold RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.15% sodium deoxycholate, 1 mM EDTA, and 0.05% SDS supplemented with a complete anti-protease cocktail from Thermofisher). 50 μg of total proteins whom concentrations have been evaluated using Pierce BCA Protein Assay Kit (23227, Thermo Scientific) were heat denaturated, loaded on a 12% SDS-PAGE, and transferred onto polyvinylidene fluoride membrane. Membranes blocked by nonfat milk were probed by a monoclonal anti-p24 antibody (NIH AIDS Reagent, ref 6521) and revealed by enhanced chemiluminescence with horseradish peroxidase-conjugated secondary antibodies on an ImageQuant LAS500 apparatus (GE Healthcare). The protein levels were standardized against GAPDH (Bio-techne, ref NB100-56875).
Virus pellets were obtained by ultracentrifugation through a 20% sucrose cushion (AH-629 rotor for 1h30 at 28,000 rpm) and resuspended in 1× PBS. The p24 concentration was determined by ELISA using Innotest HIV Antigen mAb kit (Fujirebio).
Virus pellets were obtained by ultracentrifugation through a 20% sucrose cushion (AH-629 rotor for 1h30 at 28,000 rpm) and resuspended in 1× PBS. The p24 concentration was determined by ELISA using Innotest HIV Antigen mAb kit (Fujirebio).
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