FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Methods Protoc / Enzyme-Linked Immunosorbent Assay (ELISA) Development for Eq...
Methods and protocols2025; 8(2); 37; doi: 10.3390/mps8020037

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 ODnm, 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.
Get Full Text
Publication Date: 2025-04-07 PubMed ID: 40278511PubMed Central: PMC12029847DOI: 10.3390/mps8020037Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study developed a horse-specific ELISA test to measure serum amyloid A (SAA), an inflammation marker, using antibodies generated against equine SAA proteins.
  • The new test showed high accuracy, sensitivity, and specificity, making it suitable for clinical use in diagnosing inflammation in horses.

Background and Objective

  • Serum amyloid A (SAA) is a protein that increases during inflammation and is used as a biomarker in veterinary medicine.
  • Current methods to measure horse SAA often use ELISA kits or tests designed for human SAA, which lack specificity for the horse protein.
  • The objective was to develop a species-specific ELISA capable of qualitatively and quantitatively detecting SAA specifically in horses for more accurate clinical assessment.

Methodology

  • Polyclonal antibodies were generated by immunizing mice and rabbits with recombinant equine SAA proteins to ensure specificity.
  • Serum samples were collected from two groups:
    • 32 horses exhibiting acute inflammatory disease (referred to as the SG group)
    • 25 clinically healthy horses (control group, CG)
  • For kinetic study, three horses from the SG group had their SAA levels monitored at three different time points to observe changes during disease progression.

Development and Validation of the ELISA

  • A cut-off optical density (OD) value was established at 0.06 ODnm:
    • Values ≥0.06 were considered positive for elevated SAA indicating inflammation.
    • Values <0.06 were considered negative.
  • Test performance metrics showed:
    • Sensitivity (ability to correctly identify true positives): 94%
    • Specificity (ability to correctly identify true negatives): 92%
    • Overall accuracy: 93%
    • Positive predictive value: 94%
    • Negative predictive value: 92%
  • Reproducibility was assessed:
    • Intra-assay variation (within the same test run) was 7.46% for SG and 9.63% for CG.
    • Inter-assay variation (between different test runs) was 6.1% for SG and 9.6% for CG.
  • The detection limit (lowest measurable concentration) was 0.067 ODnm.

Significance and Applications

  • The new ELISA demonstrated strong performance characteristics suitable for clinical use in horses, outperforming non-species-specific methods.
  • The use of recombinant equine SAA and species-specific antibodies enhances test accuracy and reduces the risk of cross-reactivity with other proteins.
  • This assay lays a foundation for:
    • Development of automated quantitative testing platforms for equine SAA.
    • Creation of semi-quantitative, species-specific field tests for rapid inflammation screening in veterinary practice.
  • Ultimately, this facilitates better disease diagnosis, monitoring, and treatment decisions in horses presenting with inflammatory conditions.

Cite This Article

APA
Souto PC, Santos MR, Orozco AMO, Bento LD, Ramirez-Lopez CJ, Girardi FM, Machado JCA, de Oliveira LL, da Fonseca LA. (2025). Enzyme-Linked Immunosorbent Assay (ELISA) Development for Equine Serum Amyloid A (SAA) Determination Using Recombinant Proteins. Methods Protoc, 8(2), 37. https://doi.org/10.3390/mps8020037

Publication

Methods and protocols
ISSN: 2409-9279
NlmUniqueID: 101720073
Country: Switzerland
Language: English
Volume: 8
Issue: 2
PII: 37

Researcher Affiliations

Souto, Pollyanna C
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Santos, Marcus R
  • Laboratory of Immunobiologics and Bacteriosis, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Orozco, Andrés M Ortega
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Bento, Lucas D
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Ramirez-Lopez, Camilo J
  • Laboratory of Animal Reproduction, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Girardi, Fabrícia M
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
Machado, Júlia C Assis
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
de Oliveira, Leandro L
  • Laboratory of Immunology and Glycobiology, Department of Biology, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.
da Fonseca, Leandro A
  • Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Universidade Federal de Viçosa, Viçosa 36570-900, Brazil.

Conflict of Interest Statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

This article includes 35 references
  1. Long A, Nolen-Walston R. Equine Inflammatory Markers in the Twenty-First Century. Vet. Clin. N. Am. Equine Pract. 2020;36:147–160.
    doi: 10.1016/j.cveq.2019.12.005pmc: PMC7135104pubmed: 32007299google scholar: lookup
  2. Eckersall P.D.. Proteins, Proteomics, and the Dysproteinemias. Clinical Biochemistry of Domestic Animals 6th ed. Academic Press; San Diego, MA, USA: 2008; pp. 117–156.
  3. Jacobsen S, Kjelgaard-Hansen M. Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses. Vet. Rec. 2008;163:385.
    doi: 10.1136/vr.163.11.327pubmed: 18791207google scholar: lookup
  4. Kiemle J, Hindenberg S, Bauer N, Roecken M. Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine. J. Vet. Diagnostic Investig. 2022;34:42–53.
    doi: 10.1177/10406387211056029pmc: PMC8688985pubmed: 34763564google scholar: lookup
  5. Witkowska—Piłaszewicz O.D., Żmigrodzka M, Winnicka A, Miśkiewicz A, Strzelec K, Cywińska A. Serum amyloid A in equine health and disease. Equine Vet. J. 2019;51:293–298.
    doi: 10.1111/evj.13062pmc: PMC7163734pubmed: 30565319google scholar: lookup
  6. Jacobsen S. Use of serum amyloid A in equine medicine and surgery. Vet. Clin. Pathol. 2022;52:8–18.
    doi: 10.1111/vcp.13195pubmed: 36336845google scholar: lookup
  7. Crowther J.R.. Systems in ELISA. ELISA Guidebook Humana Press; Totowa, NJ, USA: 2011; pp. 9–44.
  8. Karam B, Hines S, Skipper L, Pusterla N. Whole-Blood Validation of a New Point-of-care Equine Serum Amyloid A Assay. J. Equine Vet. Sci. 2020;94:103222.
    doi: 10.1016/j.jevs.2020.103222pubmed: 33077080google scholar: lookup
  9. Otsuka M, Sugiyama M, Ito T, Tsukano K, Oikawa S, Suzuki K. Diagnostic utility of measuring serum amyloid A with a latex agglutination turbidimetric immunoassay in bovine mastitis: Comparison with haptoglobin and alpha 1 acid glycoprotein. J. Vet. Med. Sci. 2021;83:329–332.
    doi: 10.1292/jvms.20-0550pmc: PMC7972874pubmed: 33390361google scholar: lookup
  10. Pepys M.B., Baltz M.L., Tennent G.A., Kent J, Ousey J, Rossdale P.D. Serum amyloid A protein (SAA) in horses: Objective measurement of the acute phase response. Equine Vet. J. 1989;21:106–109.
    doi: 10.1111/j.2042-3306.1989.tb02108.xpubmed: 2539996google scholar: lookup
  11. Cywińska A, Szarska E, Górecka R, Witkowski L, Hecold M, Bereznowski A, Schollenberger A, Winnicka A. Acute phase protein concentrations after limited distance and long distance endurance rides in horses. Res. Vet. Sci. 2012;93:1402–1406.
    doi: 10.1016/j.rvsc.2012.02.008pubmed: 22390917google scholar: lookup
  12. Aitken M.R., Stefanovski D, Southwood L.L. Serum amyloid A concentration in postoperative colic horses and its association with postoperative complications. Vet. Surg. 2019;48:143–151.
    doi: 10.1111/vsu.13133pubmed: 30499192google scholar: lookup
  13. De Cozar M, Sherlock C, Knowles E, Mair T. Serum amyloid A and plasma fibrinogen concentrations in horses following emergency exploratory celiotomy. Equine Vet. J. 2020;52:59–66.
    pubmed: 30912857
  14. Sinovich M, Villarino N.F., Singer E, Robinson C.S., Rubio-Martínez L.M. Can blood serum amyloid A concentrations in horses differentiate synovial sepsis from extrasynovial inflammation and determine response to treatment?. Vet. Rec. 2020;187:235.
    pmc: PMC7591800pubmed: 32098906
  15. Satoh M, Fujinaga T, Okumura M, Hagio M. Sandwich enzyme-linked immunosorbent assay for quantitative measurement of serum amyloid A protein in horses. Am. J. Vet. Res. 1995;181:1286–1291.
    doi: 10.2460/ajvr.1995.56.10.1286pubmed: 8928944google scholar: lookup
  16. Dunbar B.S., Schwoebel E.D.. Preparation of Polyclonal Antibodies. Methods 1990;182:663–670.
    pubmed: 2314261
  17. Jacobsen S., Kjelgaard-Hansen M., Petersen H.H., Jensen A.. Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations. Vet. J. 2006;172:315–319.
    pubmed: 15950503
  18. Nunokawa Y., Fujinaga T., Taira T., Okumura M., Yamashita K., Tsunoda N., Hagio M.. Evaluation of Serum Amyloid A Protein as an Acute-Phase Reactive Protein in Horses. J. Vet. Med. Sci. 1993;55:1011–1016.
    pubmed: 7509640
  19. Jacobsen S.. Review of Equine Acute-Phase Proteins. Equine Vet. Educ. 2007;53:230–235.
  20. Jacobsen S., Vinther A.M., Kjelgaard-Hansen M., Nielsen L.N.. Validation of an equine serum amyloid A assay with an unusually broad working range. BMC Vet. Res. 2019;15:1–9.
    pmc: PMC6923866pubmed: 31856804
  21. Petersen H.H., Nielsen J.P., Heegaard P.M.H.. Application of acute phase protein measurements in veterinary clinical chemistry. Vet. Res. 2004;35:163–187.
    pubmed: 15099494
  22. Hultén C., Tulamo R.-M., Suominen M., Burvall K., Marhaug G., Forsberg M.. A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA)—A clinically useful inflammatory marker in the horse. Vet. Immunol. Immunopathol. 1999;68:267–281.
    pubmed: 10438325
  23. Christensen M.B., Sørensen J.C., Jacobsen S., Kjelgaard-Hansen M.. Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum—A pilot study. BMC Res. Notes 2013;6:2–9.
    pmc: PMC3637563pubmed: 23590853
  24. Gileadi O.. Recombinant Protein Expression in E. coli: A Historical Perspective. Methods in Molecular Biology Springer; Berlin/Heidelberg, Germany: 2017; pp. 3–10.
    pubmed: 28470595
  25. Crowther J.R. Elisa—Theory and Pratice. 42nd ed. Humana Press; Totowa, NJ, USA: 1995.
  26. Robinson C.S., Singer E.R., Piviani M., Rubio—Martinez L.M.. Are serum amyloid A or D-lactate useful to diagnose synovial contamination or sepsis in horses?. Vet. Rec. 2017;181:425.
    pmc: PMC5738594pubmed: 28765498
  27. Stack J.D., Cousty M., Steele E., Handel I., Lechartier A., Vinardell T., David F.. Comparison of serum amyloid A measurements in equine synovial fluid with routine diagnostic methods to detect synovial infection in a clinical environment. Front. Vet. Sci. 2019;6:325.
    doi: 10.3389/fvets.2019.00325pmc: PMC6779708pubmed: 31632987google scholar: lookup
  28. Souto P.C., Fonseca L.A., Ortega Orozco A.M., Ramirez Lopez C.J., Ermita P.A.N., Carvalho Filho W.P., Girardi F.M.. Acute-Phase Proteins of Healthy Horses and Horses Naturally Affected by Colic Syndrome Journal of Equine Veterinary Science Acute-Phase Proteins of Healthy Horses and Horses Naturally Affected by Colic Syndrome. J. Equine Vet. Sci. 2019;80:1–4.
    pubmed: 31443825
  29. Murata H., Shimada N., Yoshioka M.. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J. 2004;168:28–40.
    pubmed: 15158206
  30. Behling-Kelly E., Haak C.E., Carney P., Waffle J., Eaton K., Goggs R.. Acute phase protein response and changes in lipoprotein particle size in dogs with systemic inflammatory response syndrome. J. Vet. Intern. Med. 2022;36:993–1004.
    pmc: PMC9151453pubmed: 35420224
  31. Christensen M., Jacobsen S., Ichiyanagi T., Kjelgaard-Hansen M.. Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA. Vet. J. 2012;194:332–337.
    doi: 10.1016/j.tvjl.2012.05.007pubmed: 22704135google scholar: lookup
  32. Andrade A.L.S.S., Zicker F.. Métodos de investigação epidemiológica em doenças transmissíveis. Volume I. Organização Pan-americana da Saúde Fundação Nacional de Saúde/Centro Nacional de Epidemiologia; Washington, DC, USA: 1997; p. 129.
  33. McDonald K.A.. Heterologous Protein Expression. Comprehensive Biotechnology In: Moo-Young M., editor. 2nd ed. Academic Press; Cambridge, MA, USA: 2011; pp. 441–449.
  34. Schwartz D., Pusterla N., Jacobsen S., Christopher M.M.. Analytical validation of a new point-of-care assay for serum amyloid A in horses. Equine Vet. J. 2018;50:678–683.
    pubmed: 29344980
  35. Kaneko J.J., Harvey J.W., Bruss M.L. Clinical Biochemistry of Domestic Animals. Academic Press; Cambridge, MA, USA: 2008.

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,093 horse owners like you who receive our email updates on horse health and nutrition.