Biology Reference
In-Depth Information
of tumor size after intravenous or intratumoral administration of
the oncolytic adenovirus is commonly used to defi ne potency.
Histology combined with quantitative information of virus DNA
content and distribution of the virus in the tumor (protein stain-
ing) are used to correlate antitumor activity with biological param-
eters such as the amount of virus that reaches the tumor (targeting),
the amount of virus replication and spread, and the blocking effect
of stroma barriers and immune responses. Paraffi n embedding pre-
serves better the morphology of tissues but if antibody fails to
detect the antigen, staining on frozen sections is required.
Therefore, it is recommended to keep tissues for both protocols.
Adenovirus is a very strong inducer of antibody and cellular
immune responses. We describe a quick method to measure neu-
tralizing antibodies against adenovirus. Specifi c anti-adenovirus
CTL responses have been detected in C57BL/6 mice (H2Db-
restricted) using peptides from E1a (SGPSNTPPEI) [
7
], E1b
(VNIRNCCYI) [
8
], and hexon (GGVINTETL and FAIKNLLLL)
(Gil-Hoyos unpublished data). ELISpot and intracellular cytokine
staining (ICS) are good techniques to monitor cellular immune
responses suing splenocytes isolated from treated mice. In vivo kill-
ing assays are appropriate to test these cellular immune responses
in vivo. For this assay splenocytes are incubated with the antigen or
epitope peptide and labeled with a high concentration of the fl uo-
rescent marker CFSE. These high-CFSE splenocytes and an equiv-
alent amount of splenocytes control (without antigen or
non-pulsed) labeled with low-CFSE are injected in the animals
where specifi c CTL immunity wants to be determined. The lysis of
these target cells relative to the lysis of nontarget cells indicates the
CTL activity against the antigen or the peptide.
2
Materials
2.1 Titration
1. Dulbecco's Modifi ed Eagle's Medium (DMEM) supplemented
with 10 % fetal bovine serum.
2. PBS-Ca/Mg: 10 g/L MgCl
2
Solution (2.5 g MgCl
2
hexahy-
drate in 250 mL H
2
O, fi lter sterilize). 10 g/L CaCl
2
Solution
(2.5 g CaCl
2
dihydrate in 250 mL H
2
O, fi lter sterilize). PBS
10× (sterile). In a 1 L beaker, add 100 mL PBS 10×, 10 mL
MgCl2 solution, 10 mL CaCl
2
solution, and cold autoclaved
H
2
O up to 1 L.
3. BCA protein stain: mix one part of reactive A in 50 parts of
reactive B and vortex the solution. This solution can be stored
for 24 h if required. For 96 wells: 96 wells × 200
µL/
well = 19.2 mL reactive A + B = >20 mL reactive A + 400
μ
L
reactive B.
