2.2. Preparation of pan-FMDV Antibody-Conjugated Magnetic Nanoparticles (pan-Ab–MNPs)

To prepare pan-Ab–MNPs, carboxylated MNPs (7.0 mg) were washed successively with 1 mL deionized water and 1 mL MES buffer (10 mM, pH 6.0), and collected using a magnetic separator. Then, the particles were incubated at 37 °C for 30 min with 1 mL MES buffer (10 mM, pH 6.0) containing a mixture (10 μL) of 800 mM EDC and 1 M NHS, followed by washing three times in the same buffer. Partially carboxylated MNPs were incubated with a 100 μg/mL pan-Ab solution for 1.5 h at 37 °C. Subsequently, the remaining carboxyl-activated groups were blocked by incubation with 1 mL of 1% BSA in MES buffer (10 mM, pH 6.0) for 2 h at 25 °C. Finally, the pan-Ab–MNPs were washed three times with the same buffer and stored at 4 °C for further experiments. The optimal EDC/NHS ratio for efficient antibody conjugation (see Figure S1) was determined by zeta potential measurements (Zetasizer Nano ZS, Malvern Panalytical, Malvern, UK). The conjugation of pan-Ab to the MNP surface was characterized by zeta potential measurements, UV–Vis spectroscopy (Optizen POP, Mecasys, Daejon, Korea), Bradford assays, and in vacuo Fourier transform infrared (FT-IR) spectroscopy (VERTEX 80v, Bruker, Ettlingen, Germany).