Hiv 1 p24 antigen elisa

Manufactured by ZeptoMetrix

Sourced in Germany, United States

The HIV-1 p24 Antigen ELISA is a laboratory diagnostic tool used for the detection and quantification of the HIV-1 p24 antigen in human serum or plasma samples. The assay employs the enzyme-linked immunosorbent assay (ELISA) principle to measure the presence and concentration of the target antigen.

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8 protocols using hiv 1 p24 antigen elisa

Construction of IRF7 Expression Lentiviral Vector

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To construct the IRF7 expression vector, the fragments encompassing the IRF7 sequence were synthetized chemically, and then cloned into the BamHI and AgeI sites in lentiviral vector (GV358) (Genechem, Shanghai). Plasmid DNA was transformed in MAX Efficiency® DH5a™ Competent Cells (Invitrogen, Mount Waverley, Victoria, Australia) as described by the manufacturer. Positive colonies were further cultured for plasmid amplification and purification using the Wizard® Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer's instructions. Purified plasmid DNA was verified for the presence of IRF7 insert by restriction digest with BamHI/AgeI. All constructs were further confirmed by DNA sequencing analysis.
293T cells were cultured at 10cm culture dishes for 24h, before the cells were co-transfected with GV358-IRF7 DNA (20μg), pHelper 1.0 (15μg), and pHelper 2.0 (15μg) using Lipofectamine 2000 in 1ml volume for 6hr (Invitrogen). After co-transfection for 6hr, the transfected supernatants were removed, and the dished were washed once by using 10ml 1×PBS, and added new cell medium. The culture supernatants were collected after the 293T cells cultured for 48hr and used as virus stock after concentration. Viral titer was determined by HIV-1 P24 Antigen ELISA (ZeptoMetrix Corporation) after infection.

Li Z., Huang Q., Chen H., Lin Z., Zhao M, & Jiang Z. (2017). Interferon Regulatory Factor 7 Promoted Glioblastoma Progression and Stemness by Modulating IL-6 Expression in Microglia. Journal of Cancer, 8(2), 207-219.

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Cell Culture and Viral Particle Quantification

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Gelatin, pronase, glycine, polybrene, and glutaraldehyde were purchased from Sigma Chemical Co. (St. Louis, MO). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA). All cell culture media and reagents were from Gibco BRL (Grand Island, NY, USA) or Hyclone (Logan, UT, USA). Viral particle concentrations were determined using HIV-1 p24 Antigen ELISA (ZeptoMetrix, Franklin, MA). Fischer F344 rats were purchased from the National Laboratory Animal Center (NLAC) (Taipei, Taiwan). Animal protocols were approved by the Animal Ethics Committee of I-Shou University.

Liu C.W., Chang L.C., Lin K.J., Yu T.J., Tsai C.C., Wang H.K, & Tsai T.R. (2014). Preparation and Characterization of Gelatin-Based Mucoadhesive Nanocomposites as Intravesical Gene Delivery Scaffolds. BioMed Research International, 2014, 473823.

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Hydrogel-Mediated Lentivirus Release

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The role of hydrogels as sustained release scaffold was performed by incubating hydrogels containing lentivirus with 293T cells in serum-containing media at 37°C for 0, 1, 3, and 5 days. Active infectious virus released from hydrogel resulted in more lentivirus particles produced from 293T cells, and lentivirus associated p24 protein can then be harvested from supernatant of culture medium. One hundred microliters of supernatant of culture medium was collected at the indicated time points and stored at −80°C until the sample concentration was determined. Hydrogels were degraded in pronase solution (Sigma Aldrich, St. Louis, MO, 1 mg/mL) to isolate the remaining virus from the gels. Viral particle concentrations were determined using an HIV-1 p24 Antigen ELISA (ZeptoMetrix, Franklin, MA) [24 (link)].

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ELISA-based Lentivirus Quantification

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Physical titers of the vectors produced were determined by quantifying the p24 HIV-1 core protein by ELISA (QuickTiter HIV Lentivirus Quantitation Kit, BioCat, Heidelberg, Germany, for RG-IDLV; and HIV-1 p24 Antigen ELISA, ZeptoMetrix, Buffalo, NY, USA, for GL-IDLV) according to the manufacturer’s instructions.

Bialek-Waldmann J.K., Domning S., Esser R., Glienke W., Mertens M., Aleksandrova K., Arseniev L., Kumar S., Schneider A., Koenig J., Theobald S.J., Tsay H.C., Cornelius A.D., Bonifacius A., Eiz-Vesper B., Figueiredo C., Schaudien D., Talbot S.R., Bleich A., Spineli L.M., von Kaisenberg C., Clark C., Blasczyk R., Heuser M., Ganser A., Köhl U., Farzaneh F, & Stripecke R. (2021). Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL. Molecular Therapy. Methods & Clinical Development, 21, 621-641.

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HIV-1 p24 Antigen ELISA Protocol

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HIV-1 p24 antigen ELISA (ZeptoMetrix, Buffalo, NY) was performed on cell culture supernatants according to manufacturer’s protocol and read on an EL800 plate reader (BioTek, Winooski, VT).

Cummins N.W., Sainski A.M., Natesampillai S., Bren G.D, & Badley A.D. (2014). Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells. Molecular and cellular therapies, 2, 1.

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Lentiviral Transduction of HT29/c1 Cells

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The initial 82 shRNA clones (shown in Supplementary Table 1) were provided by the High Throughput Biology Center (School of Medicine, JHU). The viral particles were packaged in 293T cells using Mission Lentiviral Packaging Mix (SHP001, Sigma Aldrich). The viral titers were determined by HIV-1p24 Antigen ELISA (0801111, ZeptoMetrix Corporation). The additional lentiviral transduction particles were purchased from Sigma Aldrich as GPR35: clone 1 (TRCN0000357167), clone 2 (TRCN0000357166), clone 3 (TRCN0000367796), clone 4 (TRCN0000008889), clone 5 (TRCN0000008891), clone 6 (TRCN0000008890), and beta-arrestin: clone β-arr1 (TRCN0000230149) and β-arr2 (TRCN0000280685). HT29/c1 cells grown on 96-well or 6-well plates were infected for 18–20 h at 37 °C with 5% CO₂ with a multiplicity of infection (MOI) between 10⁴ and 10⁶ viral particles. The medium was refreshed with DMEM containing 10% FCS and again incubated for 2–4 days before screening for BFT-induced morphological change. Stably transfected HT29/c1 cells were selected using DMEM medium containing 2 mg/ml puromycin.

Boleij A., Fathi P., Dalton W., Park B., Wu X., Huso D., Allen J., Besharati S., Anders R.A., Housseau F., Mackenzie A.E., Jenkins L., Milligan G., Wu S, & Sears C.L. (2021). G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis. Communications Biology, 4, 585.

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Quantifying TNFα and HIV-1 p24 Levels

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TNFα measurements of cell-free macrophage cultured supernatants were determined by ELISA (R&D Systems, Minneapolis, Minnesota) according to the manufacturer's instructions, and absorbance was measured at 450 nm using an Emax ELISA plate reader with multi-point data analysis using SoftMax Pro software (Molecular Devices, Sunnyvale, California). The detection limit for TNFα is 15.6 pg/mL. HIV-1 p24 antigen ELISA was from Zeptometrix (Franklin, Massachusetts). All measurements were performed in duplicate, and mean values of four measurements were used for statistical analysis.

Bernard M.A., Zhao H., Yue S.C., Anandaiah A., Koziel H, & Tachado S.D. (2014). Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway. PLoS ONE, 9(9), e106006.

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Lentivirus Release Kinetics Measurement

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Release studies were performed by incubation of lentivirus in serum containing media at 37 °C in 293T cells, with samples (100 μL) collected via replacement at the indicated time points and stored at −80 °C until the sample concentration was determined. Viral particle concentrations were determined using HIV-1 p24 Antigen ELISA (ZeptoMetrix, Franklin, MA, USA) [24 (link)].

Liu C.W., Chen P.H., Yu T.J., Lin K.J, & Chang L.C. (2022). WWOX Modulates ROS-Dependent Senescence in Bladder Cancer. Molecules, 27(21), 7388.

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