Simple and High-Throughput Quantification of Mono- and Bivalent Foot-and-Mouth Disease Virus Vaccine Antigens by Differential Scanning Fluorimetry

Background/Objectives: An accurate quantification of the effective antigens from different serotypes is essential for the quality control of multivalent vaccines, but it remains challenging. Herein, we developed a simple and high-throughput method using differential scanning fluorimetry (DSF) for quantifying foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent vaccines. Methods: Purified serotypes A and O FMDV were used to establish and validate the method. The DSF parameters, including the dye concentration, thermal scanning velocity, and PCR tube material, were optimized at different FMDV concentrations. The established DSF method was validated for the quantification of monovalent and A/O bivalent FMDV, and was compared with the ultracentrifugation of 86 samples from different processing stages and serotypes. Results: The DSF showed that the melting temperature (Tm) of type A (56.2 °C) was significantly higher than that of type O FMDV (50.5 °C), indicating that their Tm can be distinguished in bivalent antigens. After optimizing the DSF parameters, a strong correlation (R2 > 0.998) was observed between the 146S concentration and the maximum of the first derivative of the DSF fluorescence (d(RFU)/dT) for both serotypes A and O FMDV. The method demonstrated good reproducibility (RSD < 10%) and high sensitivity (limit of detection: 0.7 μg/mL). Using a multiple linear regression analysis, the simultaneous quantification of A and O FMDV in the bivalent mixtures achieved recovery rates of 82.4–105.5%, with an RSD < 10% for most of the samples. Additionally, the DSF results correlated well with the ultracentrifugation data (Pearson ρ = 0.9789), validating its accuracy and broad applicability. Conclusions: In summary, DSF represents a simple, rapid, and high-throughput tool for the quality control of monovalent and bivalent FMDV vaccines.