After washing three times with PBS-T, coverslips were incubated with the first monoclonal antibody anti Influenza A virus H9N2 subtype hemagglutinin 1:100 dilutions for 1 h at RT and incubated with the secondary antibody goat anti-mouse IgG H&L (FICT) ab6758 (Abcam) for 1 h. 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds. 0.05; **, 0.01; ***, 0.001. 2.4. FICT Assay Performance For FICT, LOD was determined as previously described [24]. LOD is the lowest analytic concentration likely to be reliably distinguished from blank. LOB = mean blank + 1.645 ? (SD blank) LOD = LOB + 1.645 ? (SD lowest concentration sample) Using A39-G10 McAb as a conjugate, the TL/CL of LOD was determined to be 5 HAU/mL for spiked H9N2 virus in distilled water (DW) (TL/CL: 3.39 1.24) (Figure 4A). With A45-D5 McAb as a conjugate, the TL/CL of LOD was 3.91, indicating that the titer of LOD was 5 HAU/mL because TL/CL of 2.5 and 5 HAU/mL were 1.68 0.65 and 5.3 0.61 (Mean SD), respectively. Raw data for Europium A39-G10 FICT and Europium A45-D5 FICT LODs are shown in Figure S2. In the RDT, LOD was determined to be 40 HAU/mL by both Au NP-A39-G10 RDT and Au NP-A45-D5 RDT for spiked H9N2 virus in DW, based on the very faint band at the TL (Figure S3A). These values indicated that the EuropiumFICT showed an 8-fold higher performance than the Au NPRDT. Open in a separate window Figure 4 Performance of Europium A39-G10 FICT and Europium A45-D5 FICT assays. (A) Detection limit of the FICT assay for target influenza A virus H9N2. Two-fold serially diluted influenza A virus H9N2 was tested in Europium A39-G10 FICT and Europium A45-D5 FICT. A fluorescence image by Europium-FICT Pipequaline is shown in the left panel. The linear range for FICT using the two Europium nanoparticleantibody conjugates were determined and the data (= 3) are shown as the mean SD. (B) The specificity of Europium A39-G10 FICT and Europium A45-D5 FICT. The specificity of the optimized FICT was evaluated using influenza A virus H1N1, H3N2, H5N3, H7N1, H7N7, and H9N2 subtypes applied in high titer (1280 HAU/mL). *, 0.05; ***, 0.001. To confirm the specificity of both FICT assays, six types of influenza A virus (H1N1, H3N2, H5N3, H7N1, H7N7, H9N2) were tested by Europium A39-G10 FICT and Europium A45-D5 FICT (Figure 4B). Both assays did not show cross-reactivity with McAbs when other subtype viruses were present at 1280 HAU/mL. Raw data for Europium A39-G10 FICT and Europium A45-D5 FICT specificities are shown in Figure S4. The FICT assay was optimized to detect the subtype H9 influenza Aspecific virus in chicken fecal samples. After preparing a series of two-fold dilutions for the spiked H9N2 virus, the chicken feces swab sample was taken using a disposable swab and immersed in the lysis buffer. Subsequently, 200 L of the sample was subjected to the FICT assay. The suitable lysis buffer volume for a fecal swab sample was optimized using different lysis buffer volumes and the most optimum lysis buffer volume (0.5 mL) was used for further fecal sample FICT assays (Figure S5). For FICT assays using fecal samples, the TL/CL of LOD was determined to be 80 HAU/mL (TL/CL: 7.49 1.44) by Europium A39-G10 FICT and 40 HAU/mL (TL/CL: 17.01 4.87) and by Pipequaline Europium A45-D5 FICT for the spiked H9N2 virus in chicken feces (Figure 5). In the RDT, LOD was calculated as 160 HAU/mL and 80 HAU/mL by Au NP A39-G10 RDT and Au NP A45-D5 RDT, respectively, based on LIMK2 the very faint band at the TL (Figure S3B). These values indicated that Europium A45-D5 FICT showed 2-fold higher performance than Europium A39-G10 FICT and the Europium FICT assay was 2-fold more sensitive to detect H9N2 virus spiked in chicken feces compare to Au NP RDT. Raw data of LODs for the Pipequaline spiked H9N2 virus in feces by Europium A39-G10 FICT and Europium A45-D5 FICT are shown in Figure.
After washing three times with PBS-T, coverslips were incubated with the first monoclonal antibody anti Influenza A virus H9N2 subtype hemagglutinin 1:100 dilutions for 1 h at RT and incubated with the secondary antibody goat anti-mouse IgG H&L (FICT) ab6758 (Abcam) for 1 h