Abts peroxidase substrate

Specific anti-LVS serum antibodies were determined by ELISA as described previously [28 (link)]. In brief, Immulon I plates were coated with 5 x 10⁶ bacteria per well of F. tularensis LVS diluted in 0.1M sodium bicarbonate solution and incubated for 2 hours at 37°C, then overnight at 4°C. The wells were then washed with PBS supplemented with 0.05% Tween 20 (PBST) and blocked with 10% bovine serum in PBS for 30 minutes at 37°C. Sera derived from vaccinated mice were serially diluted in PBST with 10% fetal bovine serum, added to each well and incubated for 90 minutes at 37°C. The plates were washed with PBST and incubated with horseradish peroxidase conjugated antibodies detecting mouse total IgG, or IgG isotypes, or IgM (Southern Biotech, Birmingham, AL) diluted 1:3000 in PBST with 10% bovine serum for 90 minutes at 37°C. The assay was developed by the addition of ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and 30 minutes’ incubation at room temperature protected from light. Optical density was read at 410 nm, reference 630 nm, with a microplate reader (Molecular Devices, Sunnyvale, CA). Endpoint titers were determined by selecting the dilution factor at which the average sample O.D. minus one standard deviation was greater than the average O.D. of the naïve sample, plus 3 standard deviations, as well as an O.D. value > 0.100.

Mice were individually pre-bled, infected with 5 CFU LVS ID, bled 30 days later, and sera prepared. Titers of specific anti-LVS serum antibodies were determined by ELISA as described previously [25 (link)]. Briefly, Immulon 1 plates were coated with live LVS, washed, and blocked with 10% calf serum. Serial dilutions of serum samples were added to coated wells. In each assay, sera from naïve mice was used as a negative control, and sera from LVS-hyperimmune mice, generated by repeated immunization of mice with LVS, was used as a positive control. Horseradish-peroxidase labeled antibodies (anti-IgM or anti-IgG that detects IgG₁, IgG_2a, IgG_2b, and IgG₃) (Southern Biotech, Birmingham, AL) were added, and ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used for color development. The end point titer was defined as the lowest dilution of serum that gave an optical density at 405 nm greater than the optical density at 405nm when three standard deviations were added to the OD value of the matched dilution of normal pre-bleed mouse serum, and also greater than 0.025 OD units. Geometric mean titers were calculated from endpoint titer values from 5 or 7 individual mice within a group.