Adenovirus Titering by Anti-Ad Hexon Staining

Protocol 1

1. Prepare serial dilutions (typically 1/10) of the stock in 96-well dishes using DMEM + 5% FBS. Final amount for each dilution in each well should be 100 mL. Range of dilutions will be selected according to the estimate concentration of the viral stock to titer.

2. Add 50 mL/well of a cell suspension at 1.106 cells/mL (50,000 cells/well) in DMEM + 5% FBS. The cell line must be chosen depending on characteristics of the viral stock to titer (HEK-293 cells are appropriate for most assays)

3. Incubate virus and cells for 24–48 h (time must be chosen depending on rate of replication of Ad in the cell line in order to avoid secondary infections). For HEK-293 cells incubate
for 24–36 h.

4. Remove medium from the wells very carefully to avoid cell loss. Air-dry for 3–5 min.

5. Add 100 uL of 100% ice-cold methanol to each well (fixation).

Incubate for 10 min at −20°C.

6. Aspirate methanol. Wash each well containing the cells twice with 100 uL of PBS Ca++/Mg++ 1% BSA (PBS without Ca++/Mg++ can also be used, but adding bivalent ions can prevent cells detaching from wells).

7. Add 50 uL of primary antibody diluted in PBS Ca++/Mg++ 1% BSA to each well. For most hybridomas 1/5 dilution from supernatant is recommended. If using a purified anti-adenovirus or anti-hexon Ab, 1/500 dilution can be initially tested.

Avoid bubble formation. Incubate for 1–2 h at 37°C.

8. Wash each well (3×) with 100 uL of PBS Ca++/Mg++ 1% BSA.

9. Add 50 uL of FITC or Alexa488-conjugated secondary antibody diluted in PBS Ca++/Mg++ 1% BSA (1/300 dilution is suitable for most commercial antibodies) to each well. Using Alexa488 can increase the test sensibility. Avoid bubble formation. Incubate for 1–2 h at 37°C in the dark.

10. Wash each well (3×) with 100 uL of PBS Ca++/Mg++ 1% BSA.

11. Quantify green cells in each well using an inverted fluorescence microscope. A cloud of positive cells (“comet effect”) suggests secondary infections and consequently it should be quantified as a single positive cell. For each stock, the mean of different dilutions will be used to calculate concentration.

Note that only 100 uL of each dilution has been used for analysis, so a tenfold factor should be applied to obtain transducing units per milliliter (TU/mL).

Protocol 2

Clontech Adeno-X Rapid Titer Kit

1. Split 293 cells into one 12 well plate, grow cells overnight ( 80% confluence)
2. Make 10 fold serial dilutions 10-fold serial dilutions (10^-2 ~ 10^-8 GC/mL) of purified adenoviral vector using PBS or DMEM10 as diluent.
3. Aspirate media from cells and replace with 1 mL DMEM10.
4. Add 100 ÎŒL of adenoviral dilutions 10^-6, 10^-7 10^-8 dropwise to each well.
Note: Each dilution of virus should be assayed in duplicate to ensure accuracy.
5. Incubate cells in the 37oC/5% CO2 incubator for 48 hours.
6 Aspirate medium and allow cells to dry in Biological Safety Cabinet for 5 minutes.
7. Fix cells by very gently adding 1 mL ice-cold methanol to each well.
8. Incubate the plate at -20°C for 10 minutes.
9. Aspirate methanol and gently rinse wells three times with 1 mL PBS with 1% BSA.
10. Dilute mouse anti-hexon antibody 1:1000 in PBS/BSA.
11. Aspirate final rinse from wells and add 0.5 mL of diluted mouse anti-hexon antibody to each well.
12. Incubate for 1 hour at 37oC.
13. Aspirate mouse anti-hexon antibody and gently rinse wells three times with 1 mL PBS/BSA.
14. Dilute rat-anti mouse antibody (HRP conjugate) 1:500 in PBS/BSA.
15. Aspirate final rinse from wells and add 0.5 mL of diluted rat anti-mouse (HRP conjugate) to each well.
16. Incubate for 1 hour at 37oC.
17. Dilute 10X Diaminobenzidine substrate 1:10 with 1X Stable Peroxidase Buffer. Prepare enough for 0.5 mL/well.
18. Allow 1X Diaminobenzidine solution to come to room temperature.
Note: Do not allow the 10X DAB substrate to come to room temperature.
19. Aspirate Rat Anti-Mouse (HRP conjugate) and gently rinse each well three times with 1 mL PBS/BSA.

Color Development
20. Aspirate final rinse from wells and add 0.5 mL 1X DAB solution to each well.
21. Incubate at room temperature for 10 minutes.
22. Aspirate 1X DAB solution and add 1 mL PBS to each well.

23. Count a minimum of three fields of brown/black cells using a microscope with a 20X objective.
24. Calculate infectious units (ifu)/mL for each well using the following equation:

(brown cells/field) X (fields/well)/(volume of virus [mL]) X (dilution factor)