( sub3 in 100 ml distilled water). ...)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 IgG_2a-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-IgG_2a 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 IgG_2a 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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