Standards, controls, and test samples including serum samples collected from SN1-infected and control mice were run in duplicate on precast 4 to 12% gradient bis-tris SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen) under reducing conditions at a constant current of 200 V per gel for approximately 25 min using a bolt electrophoresis mini cell apparatus (Invitrogen). One gel was stained with Coomassie brilliant blue (Coomassie Brilliant Blue R-250, Bio-Rad), and the other was transferred to nitrocellulose membrane (Novex) at 10-V constant for 60 min. After transfer, the membrane was blocked with blocking buffer (5% skim milk in PBS) for 60 min, followed by incubation for 1 hour at room temperature with specific monoclonal antibodies (anti-rabbit SpeC IgG, Toxin Technology). After excessive washing in PBS with 0.5% Tween 20, the membrane was incubated with sheep anti-rabbit IgG alkaline phosphatase antibody (Sigma-Aldrich) for 1 hour at room temperature. Following further washes, the blots were developed using SIGMAFAST BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate–nitro blue tetrazolium) (Sigma-Aldrich) according to the manufacturer’s instructions. The reaction was stopped when additional bands cease to develop.

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