Sensitive double antibody sandwich ELISA for the quantification of phosvitin

Figures & data

Figure 1. Experimental flowchart of the development of PV ELISA detection system.

Figure 2. Diagram of the detection steps of DAS-ELISA (A); and biotinylated DAS-ELISA (B).

Figure 3. The anti-PV IgY antibody levels in the WSF of egg yolk (at 1:1000 dilution) measured by Indirect ELISA and expressed as absorbance at 405 nm. Values are the means of quadruple samples. Vertical bars indicate the standard deviation. X axis indicates the weeks of immunization. Arrows indicate immunization time points.

Table 1. Concentration of total IgY and specific IgY purified from the hyperimmunized and non-immunized egg yolks.

Egg yolks	Content (mg/mL)
	Total IgY^a	Anti-PV-specific IgY
Hyperimmunized	9.0 ± 1.1	0.56 ± 0.04
Non-immunized	8.2 ± 1.1	N/A

Note: Values are the mean of quadruple samples ± SD.

^a No significant difference between total IgY content in hyperimmunized and non-immunized egg yolks.

Figure 4. SDS-PAGE analysis of Sigma PV (A); Western blot of Sigma PV developed by anti-PV IgY as primary antibody (B); Western blot of Sigma PV developed by non-specific IgY as primary antibody (C, negative control). Lane 1: standard molecular marker; Lane 2: Sigma PV.

Figure 5. Standard curve for PV detection by DAS-ELISA (A); biotinylated DAS-ELISA (B).

Table 2. Recovery (%) of PV from saline solution sample spiked with 1–100 µg/mL Sigma PV.^a

ELISA method	Recovery (%) of sigma PV spiked in µg/mL
	1	10	50	100
DAS-ELISA	80 ± 17	90 ± 15	92 + 14	92 + 11
Biotinylated ELISA	88 ± 14	92 ± 12	94 + 10	97 ± 8

^aValues represent the average of three spiking experiments and are reported as mean ± SD.

Table 3. Intra- and inter-assay variances (% CV) of biotinylated DAS-ELISA for PV quantification in five different saline solutions.

Samples	PV (µg/mL)	Intra-assay variance (% CV)	Inter-assay variance (% CV)
Saline solution 1	2.25	7	9
Saline solution 2	4.5	8	11
Saline solution 3	0.05	6	8
Saline solution 4	18	8	10
Saline solution 5	68.9	9	11