Biology Reference
In-Depth Information
Estimation of Binding Abilities of Selected Phages
The avian influenza viruses were coated (5 or 10 μg/ml; 200 μl) on a microtiter plate
with TBS buffer overnight at 4°C. The excess target was removed and blocked with
blocking buffer (milk diluent KPL, USA) for 2 hr at 4°C. The plate was then washed
with 1× TBST (TBS and 0.5% [v/v] Tween 20). Selected phages were added into the
well at the concentration of either 10
12
pfu/ml or 10
11
pfu/ml and incubated for 2 hr
at room temperature. The plate was again washed 6 times with 1× TBST. The HRP-
conjugated anti-M13 antibody (Pharmacia, USA) was diluted into 1:5,000 with block-
ing buffer and added 200 μl into each well, incubated at room temperature for 1 hr
with agitation. It was then washed six times with 1 × TBST as explained above. 200 μl
substrate solutions (22 mg ABTS in 100 ml of 50 mM sodium citrate and 36 μl of 30%
H
2
O
2
, pH 4.0) was added to each well and incubated for 60 min. Then the plate was
read using a microplate reader (Model 550, BioRad, California, USA) at 405-415 nm.
Table 4.
Peptides used in this study.
Name of the peptide
Sequence of the peptide
L-P I (Linear Peptide)
NDFRSKT
C-PI (Cyclic Peptide)
CNDFRSKTC
Control Peptide
CSWGEYDMC
Peptides
Peptides were synthesized at GL Biochem, Shanghai, China with more than 98% pu-
rity. The peptides contained the sequences as mentioned in Table 4.
Cytotoxicity Test by MTT Assay
MDCK cells (~5,000 cells/well) were grown on 96 well plates for 24 hr. The media
was replaced by serially diluted peptides or fusion phages and incubated again for
48 hr. The culture medium was removed and 25 μl of MTT (3-(4,5-dimethylthiozol-
2-yl)-3,5-dipheryl tetrazolium bromide) (Sigma) was added and incubated at 37°C for
5 hr. Then 50 μl of DMSO was added to solubilized the formazan crystals and incu-
bated for 30 mn. The optical density was measured at 540 nm in an microplate reader
(Model 550, BioRad, USA).
Virus Yield Reduction Assay in Egg Allantoic Fluid
The avian influenza A/Chicken/Iran/16/2000 (H9N2) virus suspension contain-
ing 8 or 16 HAU/50 μl was mixed with various concentrations of linear/cyclic
peptides or fusion phages (50 μl) for 1 hr at room temperature. This mixture was
then injected into the allantoic cavity of 9 day-old embryonated chicken eggs and
incubated at 37°C for 3 days. After incubation, the eggs were chilled for 5 hr, the
allantoic fluids were harvested and titrated by hemagglutination (HA) assay. As
control, virus mixed with nonspecific peptides or wild phages were injected into
the eggs.
