Development and Optimization of an Indirect Sandwich ELISA for Detection of Foot-And-Mouth Disease Virus Serotype O

Foot-and-Mouth Disease (FMD) is caused by the FMD virus. Indirect Sandwich Enzyme-Linked Immunosorbent Assay (IS-ELISA) was standardized to characterize the FMD serotype “O” virus. Total protein content in the guinea pig serum (whole serum), ammonium sulfate precipitated guinea pig serum (ASPGPS) protein and ion-exchange-based purified guinea pig serum (IEGPS) protein was measured as 52 µg/mL, 24 µg/mL and 10 µg/mL respectively. The whole serum of guinea pigs and rabbits showed the 1:32 and 1:64 anti-FMD serotype “O” virus neutralizing antibody titers, while the anti-FMD serotype “O” virus neutralizing antibody titer was 1:128 in the IEGPS proteins. IEGPS protein with 1:128 neutralizing antibody titers were used as capture/trapping antibodies in the standardization of the assay. The IEGPS protein 1:1000 diluted with 10 µg/mL of protein content was found to be optimum for capture/trapping antibodies. To cover residual blank spaces, different available blocking buffers were evaluated and Skimmed Milk Solution 5% in Phosphate-Buffered Saline (PBS5%) proved best amongst blocking buffers. Coating of 1:1000 diluted IEGPS at 37 °C for 1 h followed by storage at 4 °C for overnight was best for incubation time. FMD serotype “O” virus 1:100 diluted was optimum in IS-ELISA. Similarly rabbit anti-FMD serotype “O” virus specific immune serum 1:10,000 diluted and goat anti-rabbit IgG horseradish peroxidase conjugate 1:4000 diluted were found to be optimum during the standardization of the assay. Lastly ELISA plates proved to be best amongst the available plates for assay. In each experiment, the plateau region, test background and plate background were recorded. Lastly it became possible for the establishment of an optimized and potentially cost-effective IS-ELISA requiring further diagnostic validation in research and diagnostic laboratories in the country.