2.2.1. Immunoglobulin A

Immunoglobulin A was quantified in Asian elephant saliva by enzyme immunoassay (EIA) as described by Edwards et al. [24] with some modifications. A polyclonal rabbit anti-human IgA antibody (A0262, Dako, Glostrup, Denmark) was diluted to a working concentration of 1 mg/<sup>L</sup> in phosphate buffered saline (0.01 M phosphate buffer, 0.15 M NaCl, pH 7.2) (PBS) and 100 μL added per well to a 96-well microtiter plate (Nunc-Immuno maxisorp, Thermo Fisher Scientific, Roskilde, Denmark). After incubation overnight at 4 ◦C, plates were aspirated and washed three times with phosphate buffered saline with tween (PBS-T). Standards (0.39–100 μg/L; I2636, Sigma Aldrich, St. Louis, MO, USA) and saliva samples diluted 1:100 in PBS-T were added in duplicate. Following incubation at room temperature (RT) for 2 h on a plate shaker set to 150 rpm, plates were aspirated and washed three times with PBS-T. A polyclonal rabbit anti-human IgA antibody conjugated to horseradish peroxidase (HRP; P0216, Dako, Glostrup, Denmark) was diluted 1:10,000 in PBS-T and 100 μL added per well

before incubation at room temperature (RT) for 1 h on a plate shaker set to 150 rpm. After a final wash step, 100 μL of 3,3,5,5-tetramethylbenzidine (TMB) was added per well and incubated in the dark for 10 min at RT. Finally, the reaction was stopped with 50 μL stop solution (1N HCl) and the absorbance measured at 450 nm using a microplate reader (TECAN Sunrise, Salzburg, Austria). Assay sensitivity was 3.37 ng/mL. The EIA was validated for elephant saliva by demonstrating parallelism between serial dilutions of saliva and the IgA standards (*y* = 7.8042*x* + 0.2779, *R*<sup>2</sup> = 0.986) and significant recovery of IgA added to low concentration saliva before analysis (*y* = 0.935*x* + 0.485, *R*<sup>2</sup> = 0.997). Samples were analyzed in duplicate; inter-assay coe fficient of variation (CV) was 10.49% (n = 3), and the intra-assay CV was 2.36%.
