Mice Immunization and Antibody Detection
BALB/c (H-2d) female mice, 6 to 8 weeks old and weighing 18–20 g were purchased from CENPALAB (Havana, Cuba) and used for
all in vivo studies. Groups of 22 animals were immunized with the mixture of Co.120 (10 μg) plus plasmid pIDKE2 (100 μg), obtained using
the method described by Horn9 or the method described here. Mice were injected intramuscularly at 0, 3, 7, 12, and 16 weeks in the quadriceps muscle with
100-μL final volume of 0.9% NaCl-based solution. Normal saline was administered to negative control group under similar conditions.
Blood samples were collected 17 weeks after primary immunization from retro-orbital sinus and the sera were analyzed for antibodies
(Ab) to HCV Co.120, E1.340, and E2.680 proteins.
To detect murine anti-HCV antibodies, 96-well microtiter plates (Costar, Cambridge, MA) were coated with 100 μL of recombinant
proteins Co.120, E1.340, or E2.680 (10 μg/mL) diluted in coating buffer (50 mM carbonate buffer, pH 9.6) and incubated for
12 h at 4 °C. The wells were washed three times with 0.05% v/v Tween 20 in phosphate buffered saline pH 7.5 (PBST) and blocked with 200
μl of PBST containing 1% w/v skim milk (Oxoid Ltd, England) for 1 h at 28 °C. After three washes with PBST, 100 μL of serial
double dilutions of individual mouse sera in PBST were added and incubated at 37 °C for 1 h. The plates were washed three
times with PBST, and 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, UK) diluted 1: 25,000 was
added at 37 °C for 1 h. Positive reactions were visualized with o-phenylenediamine (Sigma) in 0.1 M citric acid, 0.2 M NaH2PO4 pH 5.0, and 0.015% v/v hydrogen peroxide as substrate, and the reaction was stopped with 50 μL of 2.5 M H2SO4. Optical density (OD) was determined at 492 nm using a plate reader (SensIdent Scan, Merck, Ger-many).
The cut-off value to consider a positive antibody response was established as twice the mean OD 492 nm of the negative control