Quantifying anti-AvCoV antibodies via S-ELISA-Concanavalin A

Lachrymal secretion and serum samples were tested for quantification of the levels of anti-AcCoV antibodies of IgA and IgG isotypes, respectively, using the S-ELISA-Concanavalin A technique performed according to Bronzoni et al. [35 (link)]. Briefly, single dilutions of 1:25 or 1:50 in PBS of lachrymal or serum samples, respectively, were tested in duplicate, and the remaining reagents, such as Concanavalin A, AvCoV IBV/PR05 antigen suspension, anti-chicken IgA, and anti-chicken IgG peroxidase conjugates (Bethyl Laboratories, Montgomery, AL, USA), were used as recommended Bronzoni et al. [35 (link)] and Lopes et al. [20 (link)]. The anti-AvCoV IgA and IgG antibody levels were evaluated as sample-to-positive (S/P) values, which were calculated, following the recommendations from Bronzoni et al. [35 (link)] and Santos et al. [24 (link)], from the differences between the optical densities (ODs) of each tested lachrymal or serum samples and the ODs of negative and positive reference samples for lachrymal secretion or serum and for the presence of anti-AvCoV IgA or IgG antibodies, respectively.