Biology Reference
In-Depth Information
3.8 Neutralizing
Anti-Adenovirus
Antibody (Nab) Assay
1. Collect blood from tail vein in capillary tubes without heparin
or EDTA. Incubate 5 min at RT and spin at 10,000 ×
g
, 4 °C
to obtain the supernatant (test serum).
2. Transfer the test serum to an eppendorf and heat-inactivate it
at 56 °C for 30 min.
3. Using DMEM/5 % FBS, dilute an adenovirus from a purifi ed
stock of known physical (vp) or infectious titer to obtain
10
7
vp/mL or 5 × 10
5
iu/mL (5 × 10
5
vp/50
μ
L or
L). Each serum to be tittered (in triplicate)
requires 1.8 mL of diluted virus (
see
Note 6
).
4. In a 96-well plate add 90
2.5 × 10
4
iu/50
μ
μ
L of diluted virus to the fi rst column
of wells and 50
L to the rest except for column 12 (no virus
control). In the fi rst row or column (dilution 1/10) add 10
μ
μ
L
of the test serum to the 90
L of diluted virus. Then perform
a ½ serial dilution transferring 50
μ
L of this row to the next
row and successively until row 10. Discard the fi nal 50
μ
L
taken from row 10. Column 11 is no test serum or virus only
control. Add 50
μ
μ
L of DMEM/5 % FBS in well 12 (no virus
control).
5. Incubate the virus and test serum (containing the Nab) for 1 h
at 37 °C.
6. Prepare a cell suspension (HEK293 cells) of 10
6
cells/mL in
DMEM/5 % FBS. Add 100
L/well (100,000 cells/well).
Incubate at 37 °C for 24 h (
see
Note 7
).
7. Determine titer by anti-hexon staining as in protocol 3.1
above, from
step 4
on. The neutralizing antibody titer is the
inverse of the dilution that reduces in 50 % the titer obtained
in well 11 (without Nab) (e.g., if 50 % of titer decrease is found
in well 6, Nab titer is 320).
μ
1. Harvest the spleen of the animal to be tested (treated with
adenovirus) and leave it in a 15 mL tube in 5 mL of RPMI
medium.
2. Put the spleen in a cell strainer and mechanically disrupt the
spleen with the fl at portion of a syringe plunger until no
fragment remains. Transfer the resuspended cells to a 15 mL
tube. Rinse the cell strainer with 5 mL of RPMI and transfer to
tube. Repeat this strainer wash to harvest all cells. Discard the
strainer.
3. Centrifuge the disrupted cells at 500 ×
g
for 5-10 min.
4. Remove the supernatant and resuspend the pellet in 2 mL of
ACK lysis buffer to lyse red blood cells. Let the cell suspension
at RT for 3 min.
5. Fill the tube with RPMI and centrifuge the cells at 500 ×
g
for
5 min.
3.9 CTL Responses
by Intracellular
Cytokine Staining
