Quantitation Kit

Catalog No. ABIN2345174

Product Details

Reactivity: Adenovirus

Detection Method: Fluorometric

Application: Quantification (Q)

Brand: QuickTiter™

Sample Type: Cell Extracts, Cell Samples

Characteristics: proprietary QuickTiter™ Adenoviral Quantitation Kit does not involve cell infection, instead it specifically measures the viral nucleic acid content of purified viruses or unpurified viral supernatant sample (See Test Principle). Especially for unpurified viral supernatant, the kit is very useful for determining the supernatant titer before the purification step. The kit has detection sensitivity limit of 1 X 109 VP or 1 X 1010 VP/mL when 100 μL of adenoviral supernatant is used in the assay, which is sufficient for mid or high-titer adenovirus sample. The entire procedure takes about 45 to 60 minutes. Each kit provides sufficient quantities to perform up to 20 tests for viral samples and controls. QuickTiter™ Adenoviral Quantitation Kit provides an efficient system for rapid quantitation of adenovirus titer for both viral supernatant and purified virus. The system may be adapted to quantitation of other viral types, such as retrovirus and lentivirus.

Components:

1. QuickTiter™ Solution A : One tube - 200 μL.
2. QuickTiter™ Adenovirus Capture Solution : One tube - 1.0 mL.
3. QuickTiter™ Solution B (10X) : Two tubes - 1.8 mL each.
4. QuickTiter™ Solution C (2X) : Two tubes - 1.5 mL each
5. CyQuant® GR Dye (400X) : One tube - 50 μL.
6. QuickTiter™ Adenovirus DNA Standard : One tube - 500μL containing 100 μg/mL Adenovirus DNA Standard

Material not included:

1. Recombinant adenovirus of interest: purified or unpurified high-titer virus supernatant
2. HEK 293 cells and cell culture growth medium
3. Cell culture centrifuge
4. 0.45 μm filter
5. 1X PBS containing 10 mM MgCl2, 1 mM CaCl2
6. 1X TE (10 mM Tris, pH 7.5, 1 mM EDTA)
7. Fluorescence Plate Reader 4

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Ultra-fast 45-60 minute procedure
• Measures the viral nucleic acid content of purified adenovirus or unpurified supernatant
• Limit of detection: 10^10 viral particles/mL from 100 μL of adenoviral supernatant

Reagent Preparation:

• 1X QuickTiter™ Solution B: Prepare a 1X QuickTiter™ Solution B by diluting the provided 10X stock 1:10 in deionized water. Store the diluted solution at room temperature.
• 1X QuickTiter™ Solution C: Prepare a 1X QuickTiter™ Solution C by diluting the provided 2X stock 1:2 in deionized water. Store the diluted solution at room temperature.
• 1X CyQuant® GR Dye: Estimate the amount of 1X CyQuant® GR Dye needed based on the number of assays including adenovirus DNA standard samples. Immediately before use, prepare a 1X CyQuant® GR Dye by diluting the provided 400X stock 1:400 in 1X TE. For best results, the diluted solution should be used with 2 hrs of its preparation.

Assay Procedure:

1. When unpurified viral supernatant is used, the supernatant is clarified by passing it through a 0.45 μm sterile filter before proceed the next step.
2. Add viral sample (1 to 100 μL) to a 1.5 mL microcentrifuge tube and adjust the final volume to 1 mL with 1X PBS containing 10 mM MgCl2, 1 mM CaCl2. Note: A proper negative control MUST be included. For purified viral sample, use the same volume of buffer solution that viruses are stored. For unpurified viral supernatant, use the same volume of uninfected 293 cell lysate supernatant that has been through three freeze/thaw cycles.
3. Add 10 μL of QuickTiter™ Solution A to the assay tube and mix by inverting the tube several times. Incubate at 37 °C for 30 minutes.
4. Mix the QuickTiter™ Adenovirus Capture Solution by vortexing for 10 seconds. Quickly transfer 40 μL of the bead capture solution to the assay tube containing the viral sample. Incubate at room temperature for 10 min on an orbital shaker.
5. Spin down the beads at 2000X g for 30 seconds. Discard the supernatant and wash the beads with 750 μL of 1X QuickTiter™ Solution B. Mix by inverting the tube several times, spin down the beads and discard the supernatant.
6. Repeat the wash step once and aspirate the final wash. To remove the last bit of liquid, centrifuge the tube again at 2000X g for 30 seconds, and remove remaining supernatant with a small bore pipette tip to avoid the beads.
7. Add 20 μL of 1X QuickTiter™ Solution C, mix with the beads by vortexing for 10 seconds, spin down the beads at 12000g for 30 seconds.
8. Transfer 5 μL supernatant to a microtiter plate suitable for fluorometer. Add 95 μL of freshly prepared 1X CyQuant® GR Dye to well(s) containing the 5 μL supernatant. Read the plate with a fluorescence plate reader using a 480/520 nm filter set.
9. Calculate adenovirus virus titer based on the standard curve.

Calculation of Results:

1. Determine Viral DNA amount: 1) Calculate Net RFU (Relative Fluorescence Unit): Net RFU = RFU (viral sample) - RFU (negative control corresponding to viral sample) 2) Use the standard curve to determine the viral DNA amount of each unknown sample.
2. Calculate Viral Titer: The average genome size of an adenovirus is 40 kbp, therefore, 1 ng adenoviral DNA = (1x10-9) g / (40,000 bp x 660 g/bp) X 6 x 1023 = 2.3 x 107 VP Virus Titer (VP/mL) = Amount of adenoviral ds DNA (ng) X 2.3 x 107 VP X (20 μL/5 μL) Viral sample volume (mL) Virus Titer (VP/mL) = Amount of adenoviral ds DNA (ng) X 9.2 x 107 VP/ng Viral sample volume (mL) Examples of Ad-β Gal Titer Quantitation: Method: HEK 293 cells were infected with Ad-β Gal at 20 MOI for 48 hrs. After three freeze/thaw cycles to release ad-β Gal viruses from infected cells, 20 mL of viral supernatant was collected and filtered through a 0.45 μm sterile filter. Ad-β Gal was further purified using ViraBind™ Adenovirus Purification Kit (Cat.# VPK-100) or CsCl2 μLtracentrifugation. The adenovirus titers were determined as described in assay instructions. Sample #1: Ad-β Gal Viral Supernatant: 100 μL was used Average Net RFU = 165.7 - 22.3 = 143.4 or 51 ng of viral DNA Virus Titer (VP/mL) = 51 (ng) X 9.2 x 107 VP/ng = 4.7 X 1010 VP/mL 0.1 mL 7 RFU (520 nm) RFU (520 nm) Sample #2: Purified Ad-β Gal (ViraBind™ Kit): 50 μL was used Average RFU = 529.4 - 3.7 = 525.7 or 197 ng viral DNA Virus Titer (VP/mL) = 197 (ng) X 9.2 x 107 VP/ng = 3.6 X 1011 VP/mL 0.05 mL Sample #3: Purified Ad-β Gal (CsCl2): 2 μL was used Average RFU = 64 - 4.0 = 60 or 22 ng viral DNA Virus Titer (VP/mL) = 22 (ng) X 9.2 x 107 VP/ng = 1.0 X 1012 VP/mL 0.002 mL

Restrictions: For Research Use only

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.

Storage: 4 °C

Storage Comment: Store all kit components at 4°C until their expiration dates.

References

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Sanchez-Lugo, Perez-Trujillo, Gutierrez-Puente, Garcia-Garcia, Rodriguez-Rocha, Barboza-Quintana, Muñoz-Maldonado, Saucedo-Cardenas, de Oca-Luna, Loera-Arias: "CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis." in: Biotechnology letters, Vol. 37, Issue 4, pp. 779-85, (2015) (PubMed).

Nuche-Berenguer, Moreno, Jensen: "Elucidation of the roles of the Src kinases in pancreatic acinar cell signaling." in: Journal of cellular biochemistry, Vol. 116, Issue 1, pp. 22-36, (2014) (PubMed).

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Rho, Ah Lee, Mi Kim, Sik Lee, Jeong Jang, Kook Kim, Sun Lee, Hyun Choi, Yong Rhim, Kim: "Sensitization of vascular smooth muscle cell to TNF-alpha-mediated death in the presence of palmitate." in: Toxicology and applied pharmacology, Vol. 220, Issue 3, pp. 311-9, (2007) (PubMed).

Merten, Jiang, Feng, Kang: "Calcineurin activation is not necessary for Doxorubicin-induced hypertrophy in H9c2 embryonic rat cardiac cells: involvement of the phosphoinositide 3-kinase-Akt pathway." in: The Journal of pharmacology and experimental therapeutics, Vol. 319, Issue 2, pp. 934-40, (2006) (PubMed).

Target Details

Background: Recombinant adenoviruses have tremendous potential in both research and therapeutic applications. There are numerous advantages they provide when introducing genetic material into host cells. The permissive host cell range is very wide. The virus has been used to infect many mammalian cell types (both replicative and non-replicative) for high expression of the recombinant protein. Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome. After entering cells, the virus remains epichromosomal (i.e. does not integrate into the host chromosome so does not activate or inactivate host genes). Recently, recombinant adenoviruses have been used to deliver RNAi into cells. HEK 293 cells or their variants are used as host cells for viral amplification. Recombinant adenoviruses can be grown at high titer (1010 VP (viral particles)/mL, which can be concentrated up to 1013 VP/mL) and purified by ViraBind™ Adenoviral Purification Kit or traditional CsCl ultracentrifugation. A particular challenge in the delivery of a gene by a viral vector is the accurate measurement of virus titer. Traditionally, infectivity particles are measured in culture by a plaque-forming unit assay (PFU) that scores the number of viral plaques as a function of dilution. Others utilize antibodies that recognize adenovirus hexon proteins by immunohistochemistry staining or FACS analysis. These methods are time-consuming, require a long infection period, and suffer from a high degree of inter- assay variability and are affected by virus-cell interactions. For highly purified virus samples, an optical absorbance at 260 nm has been used to estimate the total number of virus particles. However this method can not be used in an unpurified viral supernatant, because some of the components it contains can contributes to the optical absorbance of 260 nm.