Biology Reference
In-Depth Information
Hemagglutination Inhibition Assay
The hemagglutination inhibition (HI) assay was carried out as originally explained
by Ramanujam et al. (2002) with slight modifications to evaluate the ability of the
peptides/fusion phages to inhibit the viral adsorption to target cells. Linear/Cyclic pep-
tides or fusion phages (50 μl) in serial 2-fold dilutions in PBS were mixed with equal
volume of influenza solution (8 HAU/50 μl) and incubated at room temperature for 1.5
hr. Subsequently, 50 μl of 0.8% red blood cells were added to the above mixture and
further incubated at room temperature for 45 min.
Neuraminidase Inhibition Assay
The neuraminidase inhibition assay was carried out to test the ability of the peptide
to inhibit the viral neuraminidase activity, as explained in Aymard-Hendry et al. [40]
with slight modifications. The substrate used in this experiment was neuraminlactose
rather than feutin.
Preparation of Anti-AIV Sera
Six month old New Zealand white rabbits were used for the production of polyclonal
antibodies. Rabbits were pre-bleeded before injection. 50 μg of purified virus in PBS
together with equal amount of Freund's adjuvant was injected into the rabbit subcuta-
neously. Subsequent booster injections were done with Freund's incomplete adjuvant.
Injections were done for every 4 weeks, with bleeds 7-10 days after each injection.
Antibodies were purified with Montage
®
antibody purification kits (Millipore, USA)
as instructed by the manufacturer.
Antibody-phage Competition Assay
Wells were coated with AIV subtype H9N2 (20 μg/ml; 100 μl) as the aforesaid con-
ditions of biopanning. A mixture of purified polyclonal antibodies (1:500 dilutions;
100 μl) raised against AIV sub-type H9N2 and a series of different concentrations of
phage FP-P1 (10
8
-10
12
pfu; 100 μl) were prepared in Eppendorf tubes. After block-
ing the wells, these mixtures were added and incubated at room temperature for 1 hr.
Wells were washed and bound phages were eluted and titrated. As for the positive
control, AIV coated wells were incubated with the phage without the presence of the
polyclonal antibodies.
Peptide-phage Competition Assay
The peptide-phage competition assay was performed to assay the inhibitory effects of
synthetic peptides with its phage counterparts (FP-P1). The AIV H9N2 was coated on
a multi-well plate at the aforesaid conditions of biopanning and incubated with differ-
ent concentrations of either linear of cyclic peptides (0.0001-1,000 μM) in binding
buffer for 1 hr at 4°C. After 1 hr incubation, phage FP-P1 (10
10
pfu/100 μl) was added
and incubated at 4°C for another 1 hr. Wells were then wash six times with TBST and
the bound phages were eluted and titered. (Percentage of phage binding = (number
of phage bound in the presence of peptide competitor/number of phage bound in the
absence of peptide competitor) × 100).
