An indirect enzyme-linked immunosorbent assay (ELISA) assay (40) was used to analyze sera and purified IgG. The HR2 peptide antigen was diluted in carbonate coating buffer, and then, 100 μl of this solution was added to the wells of an ELISA microplate and incubated overnight at 4°C. After the plate was washed three times with PBS containing 0.05% Tween 20 to remove unbound antigen, 150 μl of blocking buffer (3% bovine serum albumin in wash buffer) was applied to all wells for 1 hour at 37°C. After removing the blocking buffer, 100 μl of rabbit sera or IgGs diluted in 1% blocking buffer was added to each well, and the plate was incubated for an additional 1 hour at room temperature. After three washes, HRP-conjugated goat anti-rabbit IgG was diluted (1:5000) in 1% blocking buffer, added to each well (100 μl per well), and allowed to bind to the captured rabbit IgG for 1 hour at room temperature. After five additional washes, the bound conjugate was detected with 50 μl of 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) substrate, and the reaction was stopped after 8 min by the addition of 50 μl of 1 M H2SO4. The spectroscopic absorbance of each well was measured by an automated plate reader at a wavelength of 450 nm.

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