Foot-and-mouth disease (FMD) was an exceedingly infectious disease that spread to Indonesia in May 2022. A reliable diagnostic serologic test that can distinguish between infected and vaccinated animals was an important part of FMD (serotype O) control programs in affected areas in Indonesia. For this reason, a non-structural protein (NSP) serological test based on 3ABC proteins has been used. The indirect ELISA serological test requires time, skill, and specialized equipment. An alternative method that can be employed is the lateral flow assay (LFA), which offers the advantages of simplicity and portability, enabling rapid acquisition of results. The objective of this study was to validate the efficacy of a user-friendly anti-NSP antibody LFA for rapid diagnostic purposes. This was done by assessing its sensitivity and specificity in stored samples that had previously been tested using indirect ELISA. There were 32 preserved biological materials from dairy and beef cattle in three provinces in Indonesia that were examined with developed LFA. The results of each sample on LFA were compared to the ELISA result for its sensitivity and specificity according to positive and negative values on both tests. The test had a sensitivity of 95.2% and a specificity of 100%, compared to the indirect ELISA. The measured kappa value is also very good at 0.93, so LFA can be optionally used when examining anti-NSP FMD antibodies. Therefore, the LFA anti-NSP for detecting FMD is considered reliable because of its simplicity and the accuracy of the test results. Keywords: Antibody, Bovine serum, Foot and mouth disease, Indirect ELISA, Lateral flow assay
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