Table 2. Pools of Wa HRV specific porcine IgG Abs, VP6 specific and Wa HRV specific chicken egg yolk IgY Abs and control IgY Abs used for this experiment.

Pool	N° of egg yolks	Volume (lt)	Protein concentration (mg/ml)	Total IgY concentration (mg/ml)	ELISA titer	VN titer
Control IgY pool	349	1.36	10.4	9.4	256	64
Wa HRV IgY pool A	218	0.85	5.5	2.6	16,384	4096
Wa HRV IgY pool B	513	2.00	25.8	14.3	65,536	16,384
VP6 IgY pool	220	0.85	37.5	16.0	65,536	64
Wa HRV IgG pool	-	1.60	52.4	-	65,536	16,384

Twenty Lohmann Brown Classic laying hens were hyperimmunized with 10⁷ UFF/ml of Wa HRV (G1, P1A [8]) each time and 10 hens were hyperimmunized with recombinant VP6, 7 times. Crude egg yolks were diluted in distillated water in a 1∶3 ratio and precipitated using ammonium sulfate. Sow serum was also salt-precipitated. The pellets were suspended in 10% of the original volume of egg yolk or sow serum in PBS. The protein concentration was determined by spectrophotometry (Abs 280 nm; NanoDrop ND-1000, USA). The chicken IgY and porcine IgG Ab titers to Wa HRV G1P[8] were determined by ELISA and VN assays before and after sterilization by filtration (0.22-mm-pore-size membrane filter; Millipore, USA). The total IgY concentration was determined by ELISA.