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

Abstract

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.

Keywords: acute-phase; horse; immunoassay; inflammation; protein.

Figure 1

Confirmation of rSAAeq expression in E. coli BL21-CodonPlus by 15% SDS-PAGE and Western blotting at 37 °C, induced by 0.4 mM IPTG/4 h. (A) Line 1 —MW—Bio-Rad molecular weight marker; Line 2—uninduced insoluble fraction; Line 3—induced soluble fraction; Line 4—Purified rSAAeq—presence of rSAAeq suggested at approximately 14 kDa. (B) Western blotting with anti-His antibody produced in mouse—presence of rSAAeq suggested at approximately 14 kDa.

Figure 2

Standard curve of rSAAeq for quantification of equine serum amyloid A. SAA, serum amyloid A; OD, optical density.

Figure 3

Mean and standard deviation of the absorbance of positive and negative serological samples for SAAeq. SG (animals with acute inflammatory disease) and CG (healthy animals).

Figure 4

Curve ROC—analysis of the test readings performed with samples from sick and healthy animals to determine the cut-off point. Orange dot: 94% sensitivity and 92% specificity. Cut-off 0.06.

Figure 5

The applicability of SAA-ELISA in monitoring the clinical evolution of horses with acute inflammatory disease (dental fistula, hemoparasitosis, and penile laceration) evaluated individually at three time points (D0—arrival at the hospital; D3—3 days after the start of treatment; and D7—7 days after the start of treatment). Each line represents an animal.