Each well of a Costar 96-well assay plate (Corning) was first coated with 50 μl of PBS containing 0.2 μg of the appropriate antigens. The plates were incubated overnight at 4°C and then washed five times with wash buffer containing PBS and 0.05% (v/v) Tween 20. Each well was then coated with 150 μl of a blocking buffer consisting of PBS, blotting-grade blocker (20 mg ml−1; Bio-Rad), and 5% (v/v) fetal bovine serum. The plates were incubated with the blocking buffer for 1 hour at room temperature and then washed five times with wash buffer. In the mouse sample analysis, purified IgGs were diluted in the blocking buffer to a maximum concentration of 100 μg ml−1, whereas in the rabbit sample analysis, heat-inactivated plasma was diluted by 50-fold in the blocking buffer; both samples were subjected to a 10-fold dilution series. For each sample dilution, a total volume of 50 μl was added to the wells. Each plate was incubated for 1 hour at room temperature and then washed five times with wash buffer. A 1:2000 dilution of horseradish peroxidase–labeled goat anti-mouse or anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) was then made in the wash buffer, with 50 μl of this diluted secondary antibody added to each well. The plates were incubated with the secondary antibody for 1 hour at room temperature and then washed five times with wash buffer. Last, the wells were developed with 50 μl of 3,3′, 5,5;-tetramethylbenzidine (TMB) (Life Technologies) for 3 to 5 min before stopping the reaction with 50 μl of 2 N sulfuric acid. The resulting plate readouts were measured at a wavelength of 450 nm.

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