Lentivirus ELISA Kit

Catalog No. ABIN2345175

Product Details

Target: Lentivirus

Reactivity: Lentivirus

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Brand: QuickTiter™

Sample Type: Cell Samples

Analytical Method: Quantitative

Characteristics: QuickTiter™ Lentivirus Titer Kit (Lentivirus Associated HIV p24) is an enzyme immunoassay developed for detection and quantitation of the lentivirus associated HIV-1 p24 core protein only (See Assay Principle below). After forming complexes with ViraBind™ lentivirus reagents (patented technology), while free p24 remains in supernatant, the amount of lentivirus associated p24 is measured by a HIV p24 ELISA. The kit has detection sensitivity limit of 1 ng/mL HIV p24, or about 10,000 to 100,000 TU/mL VSVG-pseudotyped lentivirus samples3-5. Each kit provides sufficient reagents to perform up to 96 assays including standard curve and lentiviral samples. QuickTiter™ Lentivirus Titer Kit (Lentivirus Associated HIV p24) provides an efficient system for rapid quantitation of lentivirus titer for both viral supernatant and purified virus.

Components: Box 1 (shipped at room temperature)

1. ViraBind™ Lentivirus Reagent A (100X) : One 1.0 mL vial
2. ViraBind™ Lentivirus Reagent B (100X) : One 1.0 mL vial
3. Sample Diluent : One 50 mL bottle containing 0.5% Triton X-100
4. Anti-p24 Antibody Coated Plate : one strip well 96-well plate
5. FITC-Conjugated Anti-p24 Monoclonal Antibody : One 20 μL vial
6. HRP-Conjugated Anti-FITC Monoclonal Antibody : One 20 μL vial
7. Assay Diluent : One 50 mL bottle
8. 10X Wash Buffer : One 100 mL bottle
9. Substrate Solution : One 12 mL amber bottle
10. Stop Solution (Part. No. 310808): One 12 mL bottle

Box 2 (shipped on blue ice packs)

1. Recombinant p24 Standard : One 100 μL vial of 10 μg/mL heat inactivated recombinant HIV1 p24 antigen in TBS plus BSA

Material not included:

1. Lentiviral Sample: purified virus or unpurified viral supernatant
2. Cell Culture Medium
3. Microcentrifuge
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. Multichannel micropipette reservoir
7. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length) 4

Application Details

Comment:

• Proprietary technology separates virus-associated p24 from free p24
• Minimizes overestimation of lentivirus titer common to traditional p24 ELISA kits
• Lentivirus quantitation on a standard microplate reader
• HIV-1 p24 Standard included

Plate: Without plate

Reagent Preparation:

• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
• FITC-Conjugated Anti-HIV1 p24 Monoclonal Antibody and HRP-Conjugated Anti-FITC Monoclonal Antibody: Immediately before use dilute the FITC-conjugated antibody 1:1000 and HRP-conjugated antibody 1:1000 with Assay Diluent. Do not store diluted solutions. Preparation of Standard Curve 1. Prepare a dilution series of recombinant HIV-1 p24 antigen in the concentration range of 100 ng/mL - 1 ng/mL by diluting the p24 stock solution in Sample Diluent (Table 1). Standard Tubes Recombinant p24 Standard (μL) Sample Diluent (μL) p24 (ng/ mL) 1 10 990 100 2 500 of Tube #1 500 50 3 500 of Tube #2 500 25 4 500 of Tube #3 500 12.5 5 500 of Tube #4 500 6.25 6 500 of Tube #5 500 3.125 7 500 of Tube #6 500 1.5625 8 0 500 0 Table 1.

Assay Procedure:

1. Prepare and mix all reagents thoroughly before use.
2. Each lentiviral sample, HIV p24 standard, blank, and control medium should be assayed in duplicate.
3. Add 100 μL of inactivated lentiviral sample or p24 antigen standard to anti-p24 antibody coated plate.
4. Cover with a Plate Cover and incubate at 37 °C for at least 4 hours or 4 °C overnight.
5. Remove Plate Cover and empty wells. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
6. Add 100 μL of the diluted FITC-Conjugated Anti-p24 Monoclonal Antibody to each well.
7. Cover with a Plate Cover and incubate at room temperature for 1 hour on an orbital shaker.
8. Remove Plate Cover and empty wells. Wash the strip wells 3 times according to step 5 above.
9. Add 100 μL of the diluted HRP-Conjugated Anti-FITC Monoclonal Antibody to all wells.
10. Cover with a Plate Cover and incubate at room temperature for 1 hour on an orbital shaker.
11. Remove Plate Cover and empty wells. Wash microwell strips 3 times according to step 5 above. Proceed immediately to the next step.
12. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
13. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
14. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 6

Calculation of Results:

I. Determine Lentivirus Associated p24 Amount: Based on p24 Standard curve, calculate the Lentivirus Associated p24 amount in the initial lentivirus sample. p24 Titer (Virus associated p24, ng/mL) = p24 (ng/mL) x Dilution Factor x 0.25 mL/1.0 mL II. Lentivirus Titer Calculation There are approximately 2000 Molecules of p24 per Lentiviral Particle (LP), therefore, 1 LP contains: 2000 x 24 x 103/(6 x 1023) g of p24 = 8 x 10-5 pg of p24 or 1 ng p24 = 1.25 x 107 LPs For reasonably packaged lentivirus vector, 1 TU is about 100 to 1000 LP3-5, therefore, 106 TU/mL = 108-9 LP/mL = 8 to 80 ng/mL Note: The calculated result is the lentivirus physical titer, p24 core protein level, and it is NOT the infectious titer (TU/mL). When the infectious titer is determined, the results vary among different target cell lines or transduction methods3-5. 8 p24 (%)

Handling

Precaution of Use: Remember that you will be working with samples containing infectious virus. Follow the recommended NIH guidelines for all materials containing BSL-2 organims.

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt, aliquot and store the Recombinant HIV-1 p24 Standard at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C until their expiration dates.

References

Loperfido, Jarmin, Dastidar, Di Matteo, Perini, Moore, Nair, Samara-Kuko, Athanasopoulos, Tedesco, Dickson, Sampaolesi, VandenDriessche, Chuah: "piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts." in: Nucleic acids research, Vol. 44, Issue 2, pp. 744-60, (2016) (PubMed).

Li, Liu, Li, Sun, Xu, Xie, Zhang: "PTPRR regulates ERK dephosphorylation in depression mice model." in: Journal of affective disorders, Vol. 193, pp. 233-41, (2016) (PubMed).

Madison, Roller, Okeoma: "Human semen contains exosomes with potent anti-HIV-1 activity." in: Retrovirology, Vol. 11, pp. 102, (2015) (PubMed).

Rohrbach, Jarboe, Anderson, Trummell, Hicks, Weaver, Yang, Oster, Deshane, Steele, Siegal, Bonner, Willey: "Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity." in: International journal of oncology, Vol. 46, Issue 3, pp. 1079-88, (2015) (PubMed).

Noh, Maze, Zhao, Xiang, Wenderski, Lewis, Shen, Li, Allis: "ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 22, pp. 6820-7, (2015) (PubMed).

Solanes, Heuzé, Maurin, Bretou, Lautenschlaeger, Maiuri, Terriac, Thoulouze, Launay, Piel, Vargas, Lennon-Duménil: "Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1." in: The EMBO journal, Vol. 34, Issue 6, pp. 798-810, (2015) (PubMed).

Lan, Wen, Saleem, Mikulak, Malhotra, Skorecki, Singhal: "Vascular smooth muscle cells contribute to APOL1-induced podocyte injury in HIV milieu." in: Experimental and molecular pathology, Vol. 98, Issue 3, pp. 491-501, (2015) (PubMed).

Xu, Liu, Sun, Guo, Zhang, Liu, Li: "CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural fibrosis." in: International journal of molecular medicine, Vol. 36, Issue 1, pp. 123-9, (2015) (PubMed).

Li, Wang, Wang, Qu, Zhu, Li, Dang, Li, Gao, Peng, Pan, Wan: "Mammalian target of rapamycin overexpression antagonizes chronic hypoxia-triggered pulmonary arterial hypertension via the autophagic pathway." in: International journal of molecular medicine, Vol. 36, Issue 1, pp. 316-22, (2015) (PubMed).

Bettens, Vermeulen, Van Cauwenberghe, Heeman, Asselbergh, Robberecht, Engelborghs, Vandenbulcke, Vandenberghe, De Deyn, Cruts, Van Broeckhoven, Sleegers: "Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations." in: Molecular neurodegeneration, Vol. 10, pp. 30, (2015) (PubMed).

Li, He, Zhang, Wang, Zhang, Tan: "Connexin43-containing gap junctions potentiate extracellular Ca²⁺-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2." in: Experimental cell research, Vol. 338, Issue 1, pp. 1-9, (2015) (PubMed).

Feng, Love, Ara, Baig, Adolph, Chelico: "Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability." in: The Journal of biological chemistry, Vol. 290, Issue 45, pp. 27188-203, (2015) (PubMed).

Xu, Liu, Song, Zhang, Li, Li, Yan, Li, Xie, Zhang: "Cerebralcare Granule® attenuates cognitive impairment in rats continuously overexpressing microRNA-30e." in: Molecular medicine reports, Vol. 12, Issue 6, pp. 8032-40, (2015) (PubMed).

Chen, Qin, Baeyens, Li, Afolabi, Budatha, Tellides, Schwartz, Simons: "Endothelial-to-mesenchymal transition drives atherosclerosis progression." in: The Journal of clinical investigation, Vol. 125, Issue 12, pp. 4514-28, (2015) (PubMed).

Chabaud, Heuzé, Bretou, Vargas, Maiuri, Solanes, Maurin, Terriac, Le Berre, Lankar, Piolot, Adelstein, Zhang, Sixt, Jacobelli, Bénichou, Voituriez, Piel, Lennon-Duménil: "Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells." in: Nature communications, Vol. 6, pp. 7526, (2015) (PubMed).

Caraveo, Auluck, Whitesell, Chung, Baru, Mosharov, Yan, Ben-Johny, Soste, Picotti, Kim, Caldwell, Caldwell, Sulzer, Yue, Lindquist: "Calcineurin determines toxic versus beneficial responses to ?-synuclein." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, Issue 34, pp. E3544-52, (2014) (PubMed).

Mufarrege, Antuña, Etcheverrigaray, Kratje, Prieto: "Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production." in: Protein expression and purification, Vol. 95, pp. 50-6, (2014) (PubMed).

Fontana, Kratje, Etcheverrigaray, Prieto: "Rabies virus-like particles expressed in HEK293 cells." in: Vaccine, Vol. 32, Issue 24, pp. 2799-804, (2014) (PubMed).

Goy, Czypiorski, Altschmied, Jakob, Rabanter, Brewer, Ale-Agha, Dyballa-Rukes, Shah, Haendeler: "The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4." in: Experimental gerontology, Vol. 56, pp. 45-52, (2014) (PubMed).

Salguero, Daenthanasanmak, Münz, Raykova, Guzmán, Riese, Figueiredo, Länger, Schneider, Macke, Sundarasetty, Witte, Ganser, Stripecke: "Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 192, Issue 10, pp. 4636-47, (2014) (PubMed).

Target Details

Target: Lentivirus

Target Type: Virus

Background: Lentivirus vector based on the human immunodeficiency virus-1 (HIV-1) has become a promising vector for gene transfer studies. The advantageous feature of lentivirus vector is the ability of gene transfer and integration into dividing and non-dividing cells1-2. The pseudotyped envelope with vesicular stomatitis virus envelope G (VSV-G) protein broadens the target cell range. Lentiviral vectors have been shown to deliver genes to neurons, lymphocytes and macrophages, cell types that previous retrovirus vectors could not be used. Lentiviral vectors have also proven to be effective in transducing brain, liver, muscle, and retina in vivo without toxicity or immune responses. Recently, the lentivirus system is widely used to integrate siRNA efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo. Lentivirus particles are produced from 293T cells through transient transfection of 3 or 4 plasmids that encodes for the components of the virion. Viral medium containing viral particles produced by packaging cells within 48-72 hr can be harvested. To ensure that pseudoviral medium is viable, and to control the number of copies of integrated viral constructs per target cell, the viral titer needs to be determined before proceeding with transduction experiments. Viral titer can be determined by transduction of HT-1080 or Hela cells, and followed by antibiotic selection of stable clones. However, it takes weeks to generate sizable stable cell colonies for counting and calculating the titer results. HIV p24 ELISA has also been used in tittering lentiviral samples, but it detects both lentivirus associated p24 and free p24 generated by 293 T cells during transient transfection. Therefore, total p24 level (lentivirus p24 and free p24) can not be used to precisely determine the viral particles in lentivirus supernatant samples.