Fig.3. Working conditions optimization of S-iELISA.
(A-B) Coating antigen concentrations and serum sample dilutions. (C-D) Second antibody dilutions and sealing times. (E-F) Sealing buffers and the incubation times of antigen and serum. (G-H) The incubation times of second antibody and exposure times of TMB.
Fig.4. Determination of the cut-off value, repeatability and specificity of S-iELISA.
(A) Forty SADS-CoV-negative serum samples were tested using the S-iELISA and the mean OD450nm value of SADS-CoV-negative serum samples plus three standard deviations (SDs) was used to calculated the cut-off value. SADS-CoV-positive serum as control. (B) Six control serum samples (5 IFA-positive samples and 1 IFA-negative samples) were tested using the S-iELISA and the OD450nm values of serum samples were used to calculate the coefficient of variation (CV) to determine the intra- and inter-assay reproducibility. (C) Porcine positive sera against SADS-CoV, PEDV, PDCoV, TGEV, PCV2, CSFV, PRV, FMDV, PRRSV, and ASFV were tested using the S-iELISA and the mean OD450nm value was calculated to determine whether the sample was positive or negative based on cut-off value. SPF pig serum as control.