Fig 3.
iPCR demonstrated earlier detection of host response antibodies in B. burgdorferi-infected mice than C6 ELISA and immunoblotting. Mouse sera were collected prior to inoculation (Pre), on specific days after intradermal inoculation with 1 × 105 B. burgdorferi B31 A3 spirochetes (left), or from uninfected mice (right) over the course of 21 days. (A) Undiluted sera were analyzed for detection of B. burgdorferi IgG antibodies using iPCR. A closed-system, real-time PCR of the DNA reporter molecule was performed using a TaqMan-based fluorescent probe assay. The mean Cq background signal, determined using uninfected sera plus 3 standard deviations, was designated the call threshold for a positive detection event and indicated here as a ΔCq of 0. Data are shown as the Cq value for each sample minus the mean background Cq plus 3 standard deviations (ΔCq). Each data point represents the average of three mice, and the standard deviation between samples is shown. (B) C6 ELISA (Immunetics, Inc., Boston, MA) was performed according to the manufacturer's instructions, with the exception that the secondary antibody was peroxidase-conjugated goat anti-mouse IgM/IgG (1:5,000). The threshold absorbance for the test is indicated (horizontal broken line). Each point represents the average of three mice, and the standard deviation between samples is shown. (C) Total B. burgdorferi sonicate was separated by SDS-PAGE and analyzed by IgM/IgG immunoblotting using immune and preimmune mouse sera diluted 1:200. The positions of the protein standards depict molecular masses (in kilodaltons). Data are representative of three mice analyzed.