EGFP-fluorescence pattern measured by flow cytometry was used to determine viral reactivation in J89GFP and THP89GFP cell lines. Whereas Agp24 release measured by enzyme immunosorbent assay (ELISA, INNOTEST® HIV-Antigen mAb, Innogenetics, Belgium) was used to determine viral reactivation in ACH-2 and J1.1 cells. J89GFP and THP89GFP cells were stimulated with the indicated compounds and stained with 7AAD to assess cell viability. At least 30,000 cells were analysed by flow cytometry. The integrated mean fluorescence intensity (iMFI, percentage of EGFP expressing cells *MFI) of live cells was used as a measure of HIV reactivation and standardized with the reactivation obtained after TNF-α treatment. The 50%, 75%, 90% and 95% effective concentrations (EC50, EC75, EC90 and EC95, respectively) were determined, and synergism analysis was performed using the CalcuSyn software (Biosoft, Cambridge, UK), based on the median effect principle33 (link). The combination index (CI) of each drug combination was plotted as a function of the fractional inhibition by computer simulation; the fractional inhibition values ranged from 0.10–0.95. CI values between 0.1–0.9 indicate a synergistic effect; whereas, values between 0.9–1.1 represent an additive effect, and >1.1 represents antagonism. Each experiment was performed in triplicate.
Innotest hiv antigen mab
Manufactured by Fujirebio
Sourced in Belgium, Japan, United States
The Innotest HIV antigen mAb is a laboratory diagnostic tool used for the detection of the HIV antigen. It functions as a monoclonal antibody-based assay.
Automatically generated - may contain errors
Lab products found in correlation
Vironostika hiv uni form 2 ag ab, by bioMérieux (2 mentions)
Inno lia hiv 1 2 score, by Fujirebio (2 mentions)
Calcusyn software, by Biosoft (1 mentions)
Celltiter glo, by Promega (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Hek293t cell line, by American Type Culture Collection (1 mentions)
Biotek synergy 4 hybrid microplate reader, by Agilent Technologies (1 mentions)
Enzygnost hiv integral 4, by Siemens (1 mentions)
Anti hiv 1 p24 antibody, by Abcam (1 mentions)
Bicinchoninic acid bca protein assay, by Thermo Fisher Scientific (1 mentions)
Peroxidase affinipure donkey anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Slhv033rs, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Inno lia hiv 1 2 score assay, by Fujirebio (1 mentions)
Polybrene, by Merck Group (1 mentions)
Hiperfect, by Qiagen (1 mentions)
26 protocols using innotest hiv antigen mab
1
Quantifying HIV Reactivation in Cell Lines
2
HIV Infection Dynamics in Activated PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Activated PBMCs were pretreated for 1 hour with a range of dendrimer concentrations. Then, cells were infected during 3 hours with 100 ng/106 cells of X4-HIV-1NL4.3 or R5-HIV-1NL(AD8) corresponding to an average of multiplicity of infection of 0.1 at 4°C. HIV p24 antigen was then quantified by Ag p24gag enzyme-linked immunosorbent assay (ELISA) kit from cell lysates (INNOTEST HIV-Antigen mAb; Innogenetics, Fujirebio Europe NV, Belgium).
3
HIV Infection Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Clients’ status was determined by using the reference standard algorithm at the AIDS reference laboratory at ITM, Antwerp, Belgium (Figure 1 ) on collected plasma samples. All samples were tested by a fourth generation ELISA (Vironostika® HIV Uni-Form II Ag/Ab, bioMérieux, France) and all reactive samples were confirmed by a Line-Immunoassay (LIA, i.e. INNO-LIA™ HIV I/II Score, Innogenetics NV, Ghent, Belgium). Samples with a negative or indeterminate LIA were tested with an antigen-enzyme-immunoassay (Ag-EIA, i.e. INNOTEST HIV Antigen mAb, Innogenetics NV, Ghent, Belgium) to confirm acute infections. In the event that the LIA could not differentiate between HIV-1 and HIV-2, we used an in-house DNA PCR.
![]()
Reference algorithm at the AIDS reference laboratory at the Institute of Tropical Medicine, Antwerp, Belgium.
4
Env-Pseudotyped Virus Production and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Env-pseudotyped virus clones described here were produced by transfecting HEK 293T cells with the env-containing pNL-SacII-lacZ/env-Ren proviral vectors, as previously described [87 (link)]. For preparation of the recombinant virus populations from the plasma and CSF of patients P#J and P#B, the env sequence region encompassing gp120 and ectodomain of gp41 was amplified and then cotransfected with NheI-linearized pNL43-Δenv-Luc2 vector DNA in HEK 293T cells, as described [109 (link)]. Forty eight hours post-transfection, cell culture supernatants were harvested, clarified by centrifugation and frozen at—80 °C. The amount of p24 antigen in the supernatants was quantified using a commercially available ELISA kit (Innotest HIV antigen mAb; Innogenetics, Gent, Belgium). All of the viruses used here (including the virus populations from P#J and P#B) were confirmed to be R5-tropic in phenotypic assays using CCR5- or CXCR4-expressing U87-CD4 cells as indicator cells.
5
Anti-HIV Activity Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infectious supernatants were obtained from calcium phosphate transfection on 293T cells and used to infect cells in the presence or absence of different concentrations of 5-hydroxytyrosol. The quantification of anti-HIV activity was performed 48 h postinfection by two different parameters depending on the virus used. First, for Renilla-luciferase viruses, cell cultures were lysed with 100 μl of buffer and relative luminescence units were obtained in a luminometre (Berthold Detection Systems, Pforzheim, Germany) after the addition of substrate to cell extracts following the specifications of ‘Renilla-luciferase assay system’ (both Promega, Madison, Wisconsin, USA). Second, by measuring p24-Gag antigen amount in supernatants using a commercially available ELISA kit (Innotest-HIV-antigen mAb; Innogenetics, Zwijndrecht, Belgium) [17 (link)] for viruses not expressing Renilla-luciferase. Cell viability in treated mock-infected cells was measured in parallel with same conditions as in the antiviral assay by CellTiter Glo (Promega) assay system. Inhibitory concentrations 50% and cytotoxic concentrations 50% (CC50) were calculated using GraphPad Prism software (La Jolla, California, USA).
6
Preparation of CCR5-tropic R5-HIV-1 Viral Stock
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral stock of CCR5-tropic R5-HIV-1NL(AD8) laboratory isolate was obtained by transient transfection with pNL(AD8) plasmid (NIH AIDS Research and Reference Reagent Program, Germantown, MD, USA) into HEK- 293 T cell line (ATCC, Manassas, VA, USA). Viral stock was clarified by centrifugation and the viral titter was later evaluated using an HIV-1 p24gag enzyme-linked Immunosorbent assay (ELISA) kit (INNOTEST HIV, Antigen mAb, Innogenetics, Ghent, Belgium), as previously reported [52 (link)].
7
HIV Antibody and Antigen Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All plasma samples were tested at ITM using a fourth-generation enzyme-linked immunosorbent assay (ELISA) detecting both antibodies and antigens (Vironostika HIV Uni-Form II Ag/Ab; bioMérieux, France) followed by a line immunoassay (LIA) (i.e., INNO-LIA HIV I/II Score; Innogenetics NV, Ghent, Belgium) and an antigen-enzyme immunoassay (Ag-EIA) (i.e., Innotest HIV Antigen MAb; Innogenetics NV, Ghent, Belgium) and in-house DNA PCR when applicable, as described for Fig. 1 .
8
Viral Production in CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Healthy donor’s CD4+ T cells were activated by PHA-L (5 μg/ml) for 48 h and cultured in RPMI medium supplemented with 10% FBS and IL-2 at 20 U/ml (complete medium) for an additional 48 h before use in the VOA. To evaluate infectious viral production after platelet–CD4+ T-cell interaction, HIV-containing platelets from InRs were added to 105 activated healthy donor’s CD4+ T cells cultured in complete medium with or without 20 μg/ml polybrene to facilitate fusion of viral envelope and host cell membrane (16 (link)). The platelet:T cell ratio employed was 2:1. After 7 days of coculture, medium was replaced and freshly prepared 105 healthy donor’s activated CD4+ T cells were added to the T cells that interacted with HIV-containing platelets to improve viral propagation for an additional 7 days. At days 0, 3, 7, and 14 of the VOA assay, culture medium was collected and replaced by fresh medium. Collected media were analyzed by HIV p24 capsid protein ELISA (Innotest HIV Antigen mAb, FUJIREBIO) to measure the cumulative viral production.
9
Reactivation of HIV-1 Latency in CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Resting CD4+ T cells cultures latently infected with HIV-1 were established and then reactivated as previously described with some modifications40 (link). Briefly, purified CD4+ T cells cultivated with 29 nM CCL19 for 1–3 days were infected by spinoculation with 300 ng p24/106 cells of NL4-3 HIV-1 (NIH AIDS Reagent Program) pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) at 1200 g for 2 h, washed, and placed back in culture in complete medium alone at 5 × 106/ml cell concentration. At day 3 post-infection, latently infected CD4+ T cells were collected and seeded at 3 × 106/ml in complete medium alone or containing an HDACi (335 nM VOR, 10 nM ROM, 20 nM PAN or 100 nM ENT), 1 µM PRO, or HDACi + PRO combinations and supplemented or not with 12.5 ng/ml IL-15; also, a culture with 10 ug/ml PHA was set for maximal HIV reactivation. After 48 h of culture, cells were washed and reseeded at 2 × 106/ml in the same initial conditions; finally, the intracellular p24 accumulation in T cells was analyzed by FACS and concentration of p24 in the culture medium was measured by ELISA (INNOTEST HIV Antigen mAb; FUJIREBIO, Japan) according to manufacturer’s protocol, 18 h and 5 days after the second stimulation, respectively.
10
Quantifying HIV Antigen p24 Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total particles (TP) concentration was estimated using the ELISA kit Innotest HIV Antigen mAb (Fujirebio, Malvern, PA, USA). To determine the total amount of p24 protein, a standard curve was established using the positive control with a 150–25 pg/mL concentration range according to the manufacturer’s instructions. A ratio of 1.25 × 107 TP/ng of p24 was used to calculate the total particles titer.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!