Rapid RCA Assay Kit

Catalog No. ABIN2345183

Product Details

Reactivity: Adenovirus

Application: Colony Formation Assay (CFA), Immunoassay (IA)

Sample Type: Cell Samples

Characteristics: ViraBind™ Adenoviral Purification Kit or traditional CsCl ultracentrifugation. During adenovirus vector production, particles may be generated which are replication competent. The probability of producing replication competent adenovirus (RCA), although low, increases with each successive amplification. RCA is thought to be produced via homologous recombination. Traditionally, RCA is measured in permissive cells by a plaque-forming unit (PFU) assay that scores the number of viral plaques as a function of dilution. These methods are time-consuming (10-14 days), require a long infection period, and suffer from a high degree of inter-assay variability and are affected by virus-cell interactions. Rapid RCA Assay Kit utilizes an antibody against adenovirus hexon proteins to visualize infected cells by immunocytochemistry staining, the kit antibody against hexon protein recognizes all serotypes of adenovirus. The hexon proteins are the largest and most abundant of the structural proteins in the adenovirus capsid, and they are distributed symmetrically to form capsid facets. Rapid RCA Assay Kit provides a quick and complete system to measure the titer of replication-competent virus in your viral prep. In contrast to the 10-day infection of a classical plaque assay, the kit only requires 2-day infection. The kit provides sufficient reagents for up to 30 tests in 6- well culture plates.

Components:

1. Anti-Hexon Antibody (1000X) : One 30 μL tube.
2. Secondary Antibody, HRP Conjugate (1000X) : One 50 μL tube.
3. DAB Substrate (25X) : One 1.5 mL tube.
4. Diluent (10X) : Three 1.5 mL tubes.

Material not included:

1. Recombinant adenovirus of interest
2. A549 cells and cell culture growth medium
3. Methanol
4. 1 % BSA/PBS
5. H2O2
6. Light Microscope
7. (optional) Wild type adenovirus

Application Details

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

Comment:

• More accurate than traditional plaque-forming unit assays
• Faster results: 2.5 days vs. 10 days
• No agar overlay steps

Assay Time: 2.5 d

Reagent Preparation:

The table below is suggested for tests in 6-well plate.

• 1X Anti-Hexon antibody solution: Prepare a 1X anti-hexon antibody solution by diluting the provided 1000X Anti-Hexon antibody stock 1:1000 in 1 % BSA/PBS. Store the diluted solution on ice.
• 1X Secondary antibody solution: Prepare a 1X Secondary antibody solution by diluting the provided 1000X stock 1:1000 in 1 % BSA/PBS. Store the diluted solution on ice.
• 1X DAB working solution: Prior to use, FRESHLY prepare a 1X DAB working solution. First dilute the provided 10X Diluent to 1X with ddH2O, and add H2O2 to a final concentration of 0.01 %. Then dilute the 25X DAB stock to 1X with 1X Diluent/ H2O2 and use the 1X DAB working solution immediately. Note: When dilute 10X diluent, use ddH2O. Heavy metals in impure H2O will cause DAB precipitation. Reagents 6 tests 12 tests 24 tests 1000X Anti-hexon Antibody 6 12 μL 24 μL 1000X Secondary Antibody 6 12 μL 24 μL 25X DAB 240 μL 480 μL 960 μL Final Volume (Each Reagent) 6 mL 12 mL 24 mL Table 1. Preparation of Antibody and DAB solutions for use in a 6-well Plate. 4

Assay Procedure:

The instructions below are suggested for assays performed in 6-well plate.

I. Virus Infection

1. Harvest fresh, healthy A549 cells and resuspend cells in culture medium at 2.5 x 105 cells/mL. Seed 2 mL in each well of a 6-well plate and incubate at 37 °C, 5 % CO2 for 1 hr.
2. Dropwise add 0.5 mL of adenoviral sample (1 x 109 VPs/mL) to each well of the 6-well plate. To ensure accuracy, perform each sample in duplicate. Note: a positive control, such as wild type Ad5, should be performed simultaneously.
3. Incubate infected cells at 37 °C, 5 % CO2 for 48 hrs.

II. Immunostaining

1. Slowly remove medium from the wells by tilting the plate and aspirating from the edge, then fix infected A549 cells by gently adding 2 mL of cold methanol to each well of the 6-well assay plate. Incubate 20 minutes at -20 °C.
2. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
3. Block for 1 hr with 1 % BSA in PBS at room temperature on an orbital shaker.
4. Add 1 mL of diluted 1X anti-Hexon antibody solution to each well and incubate for 1 hr at room temperature on an orbital shaker.
5. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
6. Add 1 mL of diluted 1X Secondary antibody solution (HRP-conjugated) to each well and incubate for 1 hr at room temperature on an orbital shaker.
7. Gently wash the fixed cells five times with 1X PBS, five minutes each wash.
8. Add 1 mL of freshly diluted 1X DAB working solution to each well and incubate for 15-30 minutes at room temperature on an orbital shaker.
9. Aspirate DAB, wash once with 1X PBS and add 2 mL of 1X PBS to each well.
10. Count positive stained cells (brown) for at least ten separate fields per well using a light microscope and 10X objective.
11. Calculate the average number of positive cells per field and RCA. 5

Calculation of Results:

1. Randomly count at least 10 fields. Calculate the average number of positive cells per field.
2. Determine the number of fields per well. For most microscopes, a standard 10X objective lens with 10X eyepiece lens has a field diameter (D) of 1.8 mm, then: Area per field = 3.14 x (D/2)2 = 3.14 x 0.92 = 2.54 mm2 For a standard 6-well plate, the surface area is 9.5 cm2/well, therefore: Fields/each well of 6-well plate = 9.5 cm2/2.54 mm2 = 9.5 cm2/2.54 x 10-2 cm2 = 374 Note: If you are not sure about the field diameter of the 10X objective lens you are using or you are using objective lenses other than 10X, the field diameter can be determined by aligning with the grids of hemacytometer (Figure 3), or referring table
3. 6 Figure
4. Hemacytometer Grid Dimensions. Fields/Well Eyepiece Lenses (6-well Objective Lenses (10X) Plate) Total Field Magnification Diameter 4X 40X 5 mm 48 10X 100X 1.8 mm 374 20X 200X 0.9 mm 1494 Table
5. Field sizes of objective lenses.
6. Calculate RCA (Replication-competent Adenovirus/VP) For wild type Ad5: RCA = (average positive cells/field) x (374 fields/well) x (dilution factor) Total VPs in 0.5 mL viral sample For recombinant adenovirus: RCA = (average positive cells/field) x (374 fields/well) 0.5 x 109 VPs in 0.5 mL viral sample Calculation Examples: Sample #1: wild type Ad5 A serial of 10-fold dilutions of the wild type Ad5 (2.0 x 1012 VP/mL) were made and RCAs were determined in a 6-well plate as described in assay instruction. At 1/107 dilution, ten fields were counted and the average positive cells/field is 40 under a standard 10X objective, therefore: RCA = (average positive cells/field) x (374 fields/well) x (dilution factor) Total VPs in 0.5 mL viral sample RCA = (40/field) x (374 fields/well) x (107) = 1.5 x 1011 RCA in 1012 VP or 15 RCAs in 100VP 1.0 x 1012 VP 7 Sample #2: Recombinant Ad-β gal Ad-β gal viral stock (1.2 x 1012 VPs/mL) was diluted to 1 x 109 VPs/mL and its RCA was determined in a 6-well plate as described in assay instruction. Ten fields were counted and no positive stained cells are visible under a standard 10X objective, therefore: RCA = (average positive cells/field) x (374 fields/well) 0.5 x 109 VPs in 0.5 mL viral sample RCA = (0/filed) x (374 fields/well) = 0 RCA in 0.5 x 109 VPs or <1 RCA in 0.5 x 109 VPs 0.5 x 109 VPs Note: If there is no positive stained cell in your viral sample, it indicates that the titer of RCA is <1 RCA in 0.5 x 109 VPs in your viral prep. For detail on adenovirus RCA safety, please follow NIH guidelines.

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.

References

Dubuisson, Day, Dhurandhar: "Accurate identification of neutralizing antibodies to adenovirus Ad36, -a putative contributor of obesity in humans." in: Journal of diabetes and its complications, Vol. 29, Issue 1, pp. 83-7, (2014) (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