Anti chicken iga

The mucosal fluid and 400-fold diluted serum samples were pipetted into Nunc MaxiSorp
flat bottom 96-well plates (Thermo Fisher Scientific, Rochester, NY, USA) coated with
killed SE cells and incubated for 2 h at 37°C. Horseradish peroxidase-conjugated goat
anti-chicken IgG or anti-chicken IgA (Bethyl Laboratories, Montgomery, TX, USA) were
added, and after reacting with tetramethylbenzidine (TMB) enzyme-linked immunosorbent
assay (ELISA) substrate solution (SureBlue Reserve, KPL/SeraCare, Milford, MA, USA)
absorbance at 450 nm (A_{450 nm}) was measured in triplicate.

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.