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Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice

sub>3 in 100 ml distilled water). Tenfold serial dilutions (1:10 to 1:10,000) of antisera from the immunized mice were made in PBS, 10% FCS, 0.05% Tween 20. After the plates were washed again 5 times in the previous wash solution, 100 l of each serial dilution was added to the wells, and the plates were incubated at room temperature for 1 hr. Following five more washes, 100 l of 1:1,000 rabbit anti-mouse-HRP (Serotec Ltd, Oxford, UK) in PBS, 10% FCS, 0.05% Tween 20 was added to each well and the plates incubated at room temperature for 1 hr. Plates were then washed 5 times before adding 150 l of ABTS substrate solution and reading the absorbance at 405 nm.

ELISA to Show Antibody Subclass Following pCMV-S DNA Vaccination
ELISA was performed as described above with the exception that the rabbit anti-mouse HRP antibody was replaced with 100 l of 1:1,000 goat anti-mouse IgG1-HRP or 1:1,000 goat anti-mouse IgG2a-HRP antibodies (Serotec Ltd) in order to detect the subclass of antibody response generated.

To determine the relative affinities of the anti-IgG1 and anti-IgG2a antibodies, a 96-well plate was coated with 50 l of 1:1,000 rabbit anti-mouse immunoglobulins (Dako, Ely, UK) and stored at 4C overnight. Fifty microliters (50 l) of the control IgG1 antibody 730 and IgG2a antibody 1143B7 were then added at concentrations of 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 g/ml and the plates incubated at room temperature for 1 hr. Next, 50 l of 1:1,000 rabbit anti-mouse-HRP antibody was added and the plates incubated for 1 hr at room temperature. ABTS substrate solution (150 l) was then added and absorbance read at 405 nm.

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