Enzyme-Linked Immunosorbent Assay (ELISA) Development for Equine Serum Amyloid A (SAA) Determination Using Recombinant Proteins

We aimed to develop a species-specific ELISA for qualitatively and quantitatively determining serum amyloid A (SAA) in horses. Current methods for measuring SAA in horses utilize ELISA or immunoturbidimetric tests designed for human SAA, which are not specific to horses. Mice and rabbits were used to generate polyclonal antibodies against equine SAA. The study examined serum samples from 32 horses with acute inflammatory disease (SG) and 25 clinically healthy horses. Furthermore, the SAAeq kinetics were observed in three horses from the SG group at three different timepoints. The SAA-ELISA established a cut-off at 0.06 OD₄₉₂nm, where values equal to or higher than this were deemed positive, while values below it was considered negative. The test exhibited a sensitivity of 94% and specificity of 92%, resulting in an overall accuracy of 93%. The positive and negative predictive values were 94% and 92%, respectively. Coefficients of variation for inter- and intra-assay were 6.1% and 7.46% for SG and 9.6% and 9.63% for the control group (CG). The detection limit was determined to be 0.067. The SAA-ELISA proved its worth by demonstrating satisfactory performance, paving the way for the development of automated quantitative tests and species-specific semi-quantitative tests. This paves the way for their application in practical field settings.