Quantification of PiCV-specific IgY-SBC

The MultiScreen plates (Merck Millipore, USA) were coated with 100μL/ well of a PBS solution of PiCV rCP (conc. 20μg/ 1 mL). The samples were standardized to 1.5 x 10⁵ mononuclear cells and seeded in triplicate directly to the wells of previously coated with PiCV rCP plates. After incubation with Iscove’s modified Dulbecos medium (IMDM; Sigma Aldrich, Germany), the plates were rinsed and incubated overnight with biotinylated rabbit anti-pigeon IgY (Antibodies-online, USA). After rinsing, 100 μL of streptavidin, alkaline phosphatase (S-AP) (Vector Laboratories, USA) dissolved 1:500 in PBS (Sigma Aldrich, Germany) were added to each well. Enzymatic reaction was performed with an alkaline phosphatase substrate (BCIP/NBT; Vector Laboratories, USA) and stopped with distilled water. All details concerning rinsing and incubation steps were described in our previous study [5 (link)]. Counting of spot forming units (SFU) corresponding to anti-PiCV rCP IgY-SBC was performed with an Eli.Scan plate scanner and Eli.Analyse software (A-EL-VIS, Germany). Data were expressed as the mean absolute number of SFU +/- standard deviation per 1 x 10⁶ of mononuclear cells in each group in each day of sampling.