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Product ManualQuickTiter Adenovirus Quantitation KitCatalog NumberVPK-10620 assaysFOR RESEARCH USE ONLYNot for use in diagnostic procedures

IntroductionRecombinant adenoviruses have tremendous potential in both research and therapeutic applications.There are numerous advantages they provide when introducing genetic material into host cells. Thepermissive 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. Recombinantadenoviruses are especially useful for gene transfer and protein expression in cell lines that have lowtransfection 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. Recombinantadenoviruses can be grown at high titer (1010 VP (viral particles)/mL, which can be concentrated up to1013 VP/mL) and purified by Cell Biolabs ViraBind Adenoviral Purification Kit or traditional CsClultracentrifugation.A particular challenge in the delivery of a gene by a viral vector is the accurate measurement of virustiter. 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 thatrecognize adenovirus hexon proteins by immunohistochemistry staining or FACS analysis. Thesemethods are time-consuming, require a long infection period, and suffer from a high degree of interassay variability and are affected by virus-cell interactions. For highly purified virus samples, anoptical absorbance at 260 nm has been used to estimate the total number of virus particles. Howeverthis method can not be used in an unpurified viral supernatant, because some of the components itcontains can contributes to the optical absorbance of 260 nm.Cell Biolabs’ proprietary QuickTiter Adenoviral Quantitation Kit does not involve cell infection;instead it specifically measures the viral nucleic acid content of purified viruses or unpurified viralsupernatant sample (See Test Principle). Especially for unpurified viral supernatant, the kit is veryuseful for determining the supernatant titer before the purification step. The kit has detectionsensitivity limit of 1 X 109 VP or 1 X 1010 VP/mL when 100 µL of adenoviral supernatant is used inthe assay, which is sufficient for mid or high-titer adenovirus sample. The entire procedure takes about45 to 60 minutes. Each kit provides sufficient quantities to perform up to 20 tests for viral samples andcontrols.QuickTiter Adenoviral Quantitation Kit provides an efficient system for rapid quantitation ofadenovirus titer for both viral supernatant and purified virus.2

Assay Principle3

Related Products1. AD-100: 293AD Cell Line2. AD-200: ViraDuctin Adenovirus Transduction Reagent3. VPK-099: ViraBind Adenovirus Miniprep Kit4. VPK-109: QuickTiter Adenovirus Titer Immunoassay Kit5. VPK-110: QuickTiter Adenovirus Titer ELISA Kit6. VPK-111: Rapid RCA Assay Kit7. VPK-252: RAPAd CMV Adenoviral Expression System8. VPK-254: RAPAd CMV Adenoviral Bicistronic Expression System (GFP)Kit Components1. QuickTiter Solution A (Part No. 40020): One tube – 200 µL.2. QuickTiter Adenovirus Capture Solution (Part No. 40021): One tube – 1.0 mL.3. QuickTiter Solution B (10X) (Part No. 40022): Two tubes – 1.8 mL each.4. QuickTiter Solution C (2X) (Part No. 40023): Two tubes – 1.5 mL each5. CyQuant GR Dye (400X) (Part No. 105101): One tube – 50 µL.6. QuickTiter Adenovirus DNA Standard (Part No. 40025): One tube – 500µL containing 100µg/mL Adenovirus DNA StandardMaterials Not Supplied1. Recombinant adenovirus of interest: purified or unpurified high-titer virus supernatant2. HEK 293 cells and cell culture growth medium3. Cell culture centrifuge4. 0.45 µm filter5. 1X PBS containing 10 mM MgCl2, 1 mM CaCl26. 1X TE (10 mM Tris, pH 7.5, 1 mM EDTA)7. Fluorescence Plate ReaderStorageStore all kit components at 4ºC.Safety ConsiderationsRemember that you will be working with samples containing infectious virus. Follow therecommended NIH guidelines for all materials containing BSL-2 organisms.4

Preparation of Reagents 1X QuickTiter Solution B: Prepare a 1X QuickTiter Solution B by diluting the provided 10Xstock 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 2Xstock 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 thenumber of assays including adenovirus DNA standard samples. Immediately before use, prepare a1X CyQuant GR Dye by diluting the provided 400X stock 1:400 in 1X TE. For best results, thediluted solution should be used within 2 hrs of its preparation.Preparation of Standard Curve1. To create adenovirus DNA standards from 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 0µg/mL (1:2 serial dilution), label nine microcentrifuge tubes #1 to #9.2. Add 20 µL of 1X QuickTiter Solution C to tube #2 to #9, transfer 20 µL of 100 µg/mLQuickTiter Adenovirus DNA Standard to tube #1 and #2. Mix tube #2 well, transfer 20 µL of themixture (50 µg/mL) to the next tube. Repeat the steps through tube #8 and use tube #9 as a blank.3. Transfer 5 µL of each dilution including blank to a microtiter plate suitable for fluorometer. Add95 µL of 1X CyQuant GR Dye to each of the wells containing the 5 µL sample. Read the platewith a fluorescence plate reader using a 480/520 nm filter set.Harvesting Infected Cell LysateThe following procedure is suggested for a T75 flask and may be optimized to suit individual needs.1. Use HEK 293 cells that have been passaged regularly 2-3 times prior to the infection. Culturethese cells until the monolayer is 90-100% confluent.2. Replace the cell culture media with new growth media, 15 mL per flask. Next, the adenovirus isadded to the culture. Either crude or purified viral stock can be used. A multiplicity of 10 PFU(plague forming units) per cell is desired.3. After 24 hr, some cells should be floating. Add 10 mL growth media to the culture flask and allowthe viruses to expand for another 24 hrs. When all the cells are floating, gently shake the cultureflask several times and harvest all media, including cells, in a sterile tube.4. Release the adenoviruses from the cells with three freeze/thaw cycles. Centrifuge at 3000 rpm for10 minutes to pellet the cell debris. Discard the pellet and save supernatant. If a large amount ofcell debris is still visible, centrifuge the supernatant again.5. The viral supernatant can be stored at -80ºC or immediately used in the titration step.Assay Protocol1. When unpurified viral supernatant is used, the supernatant is clarified by passing it through a 0.45µm sterile filter before proceed the next step.5

2. Add viral sample (1 to 100 µL) to a 1.5 mL microcentrifuge tube and adjust the final volume to 1mL with 1X PBS containing 10 mM MgCl2, 1 mM CaCl2.Note: A proper negative control MUST be included. For purified viral sample, use the samevolume of buffer solution that viruses are stored. For unpurified viral supernatant, use the samevolume 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 severaltimes. Incubate at 37ºC for 30 minutes.4. Mix the QuickTiter Adenovirus Capture Solution by vortexing for 10 seconds. Quickly transfer40 µL of the bead capture solution to the assay tube containing the viral sample. Incubate at roomtemperature 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 with750 µL of 1X QuickTiter Solution B. Mix by inverting the tube several times, spin down thebeads and discard the supernatant.6. Repeat the wash step once and aspirate the final wash. To remove the last bit of liquid, centrifugethe tube again at 2000X g for 30 seconds, and remove remaining supernatant with a small borepipette tip to avoid the beads.7. Add 20 µL of 1X QuickTiter Solution C, mix with the beads by vortexing for 10 seconds, spindown the beads at 12000g for 30 seconds.8. Transfer 5 µL supernatant to a microtiter plate suitable for fluorometer. Add 95 µL of freshlyprepared 1X CyQuant GR Dye to well(s) containing the 5 µL supernatant. Read the plate with afluorescence plate reader using a 480/520 nm filter set.9. Calculate adenovirus virus titer based on the standard curve.Example of ResultsThe following figures demonstrate typical quantitation results. One should use the data below forreference only. This data should not be used to interpret actual results.6

70050454050035RFU (520 nm)RFU (520 nm)60040030020030252015101005000100200Adenoviral DNA (ng)030051015Adenoviral DNA (ng)20Figure 1: Adenovirus DNA Standard Curve. The QuickTiter Adenovirus DNA Standard wasdiluted as described in the above instructions. Fluorescence measurement was performed onSpectraMax Gemini XS Fluorometer (Molecular Device) with a 485/538 nm filter set and 530 nmcutoff.Calculation of Adenovirus Titer (VP/mL)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 VPVirus 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/ngViral 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 threefreeze/thaw cycles to release ad-β Gal viruses from infected cells, 20 mL of viral supernatantwas collected and filtered through a 0.45 µm sterile filter. Ad-β Gal was further purified usingViraBind Adenovirus Purification Kit (Cat.# VPK-100) or CsCl2 ultracentrifugation. Theadenovirus titers were determined as described in assay instructions.Sample #1: Ad-β Gal Viral Supernatant: 100 µL was usedAverage Net RFU 165.7 – 22.3 143.4 or 51 ng of viral DNAVirus Titer (VP/mL) 51 (ng) X 9.2 x 107 VP/ng 4.7 X 1010 VP/mL0.1 mLSample #2: Purified Ad-β Gal (ViraBind Kit): 50 µL was used7