Fig 1.
Extract antigen from oil emulsion vaccine.
Antigens from oil emulsion vaccines were recovered from the aqueous phases after the rupture of the water-in-oil emulsions with n-butyl alcohol. Briefly, nine volumes of vaccine were thoroughly mixed with one volume of n-butyl alcohol and centrifuged for 5 min at 4°C, 5,000 g. The lower aqueous phases were collected carefully and used for subsequent antigen quantification. (A) Vaccine No. 1. (B) Vaccine No. 1 mixed with n-butyl alcohol and breaking of the emulsion complete. (D) Vaccine No. 2. (C) Vaccine No. 2 mixed with n-butyl alcohol and breaking of the emulsion complete. (E) Vaccine No. 3. (F) Vaccine No. 3 mixed with n-butyl alcohol and breaking of the emulsion incomplete, the intermediate layer was not clear and the volume of the aqueous layer was quite small. (G) Vaccine No. 3 mixed with n-butyl alcohol complete (by increasing the amount of n-butyl alcohol).
Table 1.
FMD vaccine manufacturers.
Fig 2.
The intra-assay precision (A, 2.87–12.43%) and the inter-assay precision (B, 4.33–8.67%) of the DAS ELISA.
Ten FMDV samples, including 4 vaccine samples (V1-V4), 3 antigen samples (Ag1-Ag3) and 3 virus samples (B1-B3), were assayed 9 times to determine precision within an assay and between assays, the coefficients of variations (CV, %) were plotted and the means of the samples were showed.
Table 2.
Linearity of dilution: the measured concentration of 4 samples versus the expected concentration at certain dilutions.
Fig 3.
The specificity of the DAS ELISA.
The specificity of the DAS ELISA was analysed through dilution series of the following FMDV strain antigens: O/MYA98, O/China/99, Asia 1/JSL and A/HuBWH serotypes, from a constant high initial amount (2.0 μg/mL), and the blank samples of BHK-21 cells and PBS, as well as the other disease antigens, such as: swine vesicular disease virus (SVDV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and bovine viral diarrhoea virus (BVDV). The results indicated that the O/MYA98 and O/China/99 can be specifically detected among the others by the absorbance values obtained for testing.
Fig 4.
Comparison of the 146S antigen quantification by the DAS ELISA and SDG method.
85 FMDV samples, including FMDV live (n = 17, 39#-55#), inactivated antigen samples (n = 50,1#-20#,57#-85#) and vaccine samples (n = 18, 21#-38#), were tested with both methods. The results were compared using the software “Curve Expert 1.4”, and it demonstrated that the DAS ELISA was highly correlated with the traditional SDG method (R2 = 0.9215, P<0.01).
Table 3.
Vaccine samples tested in the ELISA comparison test.
Fig 5.
The inactivated FMDV antigen alone or mixed with n-butyl alcohol, the pH values of antigen and mixture are unchanged.