Biology Reference
In-Depth Information
3.4 Adenovirus
Characterization
The most common infectious assay involves the detection of a
cytotoxic effect on cells following viral spread. The fastest infec-
tious assay relies on the transfer of a specifi c gene coding for a
protein and the subsequent detection of this protein. Other infec-
tious assays have relied on molecular DNA detection techniques
following viral DNA replication. Here we describe a titration
method based on the detection of hexon viral protein, a specifi c
gene transferred by the adenoviral vector and expressed during its
infection cycle. This method can be applied to RCA quantifi cation
(
see
Note 12
).
3.4.1 Titration of
Recombinant Adenovirus
Using Anti-Ad/Hexon
Staining
1. The day before, seed cells in a collagen-coated 96-well plate, in
a quantity enough to obtain a confl uence of 80 % on the day of
the experiment (35,000 cells/well).
2. Prepare serial dilutions of the adenoviral vector in 24-well
plates using DMEM + 2 % FBS (
see
Note 13
).
3. Aspirate the medium of the cells (96-well plate prepared in
step 1
) and add 100
L/well of each viral dilution (
see
Note 14
).
4. Incubate virus and cells for 48 h (
see
Note 15
).
5. Remove medium from wells very carefully to avoid cell loss.
Air dry for 5-10 min.
6. Add 75
μ
μ
L/well of ice-cold methanol. Incubate for 10 min at
−20 °C.
7. Aspirate methanol. Wash cells twice with 100
μ
L/well of
D-PBS + 1 % BSA.
8. Add 50
L/well of primary Antibody diluted in D-PBS + 1 %
BSA (
see
Note 16
), avoiding bubble formation. Incubate for
1-2 h at 37 °C.
9. Wash twice with 100
μ
L/well of D-PBS + 1 % BSA.
10. Add 50 mL/well of FITC or Alexa488-conjugated secondary
antibody diluted in D-PBS + 1 % BSA, avoiding bubble forma-
tion (1/300 dilution is suitable for most commercial antibodies).
Using Alexa488 can increase test sensitivity. Incubate for 1-2 h
at 37 °C in the dark.
11. Wash twice with 100
μ
L/well of D-PBS + 1 % BSA.
12. Count green cells using an inverted fl uorescence microscope.
A cloud of positive cells (“comet effect”) is suggestive of sec-
ondary infections and, consequently, it should be counted as a
single positive cell. The mean of different dilutions will be used
to calculate the infectious titer. Note that only 100
μ
L of each
dilution is being used for analysis, so a tenfold factor should be
applied to obtain infectious units per milliliter (IU/mL).
μ
1. Dilute samples in lysis buffer (usually 1:10 or 1:20). A control
dilution with PBS Ca
2+
/Mg
2+
with 10 % glycerol should be
prepared and used as a blank for the optical density measure.
3.4.2 Quantifi cation
of Adenovirus Particles
by Spectrophotometry
