Streptavidin horseradish peroxidase conjugate

96-well nitrocellulose-bottomed microplates (Millipore, Billerica, MA) were coated with a rat antibody specific for mouse IFNγ or for mouse IL-5 (BD Pharmigen, Le Pont de Claix, France). After plate washing, freshly isolated splenocytes (2 x 10⁵ cells/well) from immunized and control BALB/c ByJ mice were added to the plate and incubated overnight with murine IL-2 (10 μg/mL; Roche-Boehringer Mannheim, Meylan, France) and either heat-inactivated HSV-2 strain G (10⁵ CCID₅₀/mL equivalents), recombinant HSV gD2 (1 μg/mL; Mybiosource, San Diego, CA), concanavalin A (2.5 μg/mL) as a positive control, or medium as a negative control. The plates were washed and the locations where cells secreting INFγ or IL-5 during incubation were stained with a biotinylated anti-mouse IFNγ or IL-5 antibody (BD Pharmigen, Le Pont de Claix, France), a streptavidin-horseradish peroxidase conjugate (Southern Biotechnology), and a chromogenic substrate (amino ethyl carbazole; Sigma-Aldrich, St. Louis, MO). Plates were read with an automatic ELISPOT reader (Microvision Instruments, France) and stained cells were counted with Spot software (Microvision Instruments, France). Results were expressed as the number of cells (spots) per 10⁶ splenocytes. The threshold of positivity was established at 20 cells/10⁶ splenocytes.

The indirect ELISA was used for determination of the mouse serum IgE and IgG1 specific to the Dp-CE. The assay was performed as described previously [50 (link)]. Each well of an ELISA plate was coated with Dp-CE (1 μg in 100 μl carbonate-bicarbonate buffer, pH 9.6) and blocked with 200 μl of 1% BSA in PBS. Triple Dp-CE-coated wells were added with individual mouse serum samples (diluted 1:4 for IgE and 1:100 for IgG1 determinations). After keeping the plates at 37°C for 1 h, all wells were washed with PBS containing 0.05% Tween-20 (PBST) and then added with 100 μl of rat anti-mouse IgE-biotin (Abcam^®, Cambridge, USA) diluted 1:3,000 in PBST for IgE detection or goat anti-mouse IgG1-biotin (Southern Biotech, Birmingham, Alabama, USA) diluted 1:10,000 for IgG1 detection. The plates were incubated at 37°C for 1 h. Streptavidin-horseradish peroxidase conjugate (Southern Biotech) and freshly prepared 2,2’-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid; ABTS) substrate (Gaithersburg, MD, USA) were used for color development. The color reaction was stopped by adding 50 μl of 1% SDS to each well. Wells added with PBS instead of the mouse serum served as blank. Optical density (OD) at 405 nm of the content in each well was determined against the blank. Data are presented as mean ± standard deviation (SD) of OD_405nm of serum samples of mice of the same group.