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Home / Equine Research Bank / Clin Vaccine Immunol / Development of an enzyme-linked immunosorbent assay using a ...
Clinical and vaccine immunology : CVI2013; 20(8); 1143-1149; doi: 10.1128/CVI.00245-13

Development of an enzyme-linked immunosorbent assay using a recombinant LigA fragment comprising repeat domains 4 to 7.5 as an antigen for diagnosis of equine leptospirosis.

Abstract: Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5-5.5), 5.5th to 6.5th (LigACon5.5-6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that LigACon4-7.5 is the best candidate diagnostic antigen in a slot-shaped dot blot assay. LigACon4-7.5 was further evaluated as an indirect enzyme-linked immunosorbent assay (ELISA) antigen for the detection of Leptospira antibodies in equine sera. This assay was evaluated with equine sera (n = 60) that were microscopic agglutination test (MAT) negative and sera (n = 220) that were MAT positive to the 5 serovars that most commonly cause equine leptospirosis. The indirect ELISA results showed that at a single serum dilution of 1:250, the sensitivity and specificity of ELISA were 80.0% and 87.2%, respectively, compared to those of MAT. In conclusion, an indirect ELISA was developed utilizing a recombinant LigA fragment comprising the 4th to 7.5th repeat domain (LigACon4-7.5) as a diagnostic antigen for equine leptospirosis. This ELISA was found to be sensitive and specific, and it yielded results that concurred with those of the standard MAT.
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Publication Date: 2013-05-29 PubMed ID: 23720368PubMed Central: PMC3754523DOI: 10.1128/CVI.00245-13Google Scholar: Lookup
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  • Evaluation Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

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.

This research aimed to develop a new technique for the diagnosis of equine leptospirosis, a common disease in horses. The researchers created and tested various fragments of a protein, LigA, and found that one particular fragment proved most effective for diagnosing the disease. An assay (test) using this protein fragment was found to be both sensitive and specific, agreeing with results from the current standard test.

Research Background

  • Leptospira Immunoglobulin-like (Lig) proteins are proteins which are found on the surface of cells and they have 630 amino acids in their N-terminal region that are conserved, meaning they remain unchanged across different species.
  • The research aimed to determine the potential of these proteins, specifically LigA, in the diagnosis of equine leptospirosis, a bacterial disease common in horses.

Creating Fragments of LigA Protein

  • For the study, the LigA protein, specifically its conserved region, was divided into seven fragments featuring different repeat domains. These proteins were labeled as LigACon1-3, LigACon4-7.5, LigACon4, LigACon4.5-5.5, LigACon5.5-6.5, LigACon4-5, and LigACon6-7.5.
  • The fragments were then used to create seven different recombinant Lig proteins.

Dot Blot Assay for Diagnosing Equine Leptospirosis

  • The recombinant Lig proteins were screened using a diagnostic tool known as a slot-shaped dot blot assay. This assay is used to detect proteins and immunoglobulins (antibodies) in a sample.
  • The results showed LigACon4-7.5 to be the most viable candidate for diagnostic antigen in the slot-shaped dot blot assay.

Evaluating LigACon4-7.5 as an ELISA Antigen

  • The LigACon4-7.5 fragment was further tested as an indirect enzyme-linked immunosorbent assay (ELISA) antigen. ELISA is a test that uses antibodies and color change to identify a substance.
  • The researchers used the ELISA to detect Leptospira antibodies in horse blood serum. They tested sera that were known to be either negative or positive for equine leptospirosis according to the current standard test, the microscopic agglutination test (MAT).
  • The results showed that the ELISA had a sensitivity of 80.0% and a specificity of 87.2% at a serum dilution of 1:250, compared to MAT.

Conclusion

  • In conclusion, the research successfully developed an indirect ELISA test for equine leptospirosis using the recombinant LigACon4-7.5 protein fragment.
  • This new test is sensitive to the bacteria causing the disease and specific to it, meaning it is unlikely to produce false positives. Further, the results obtained using this ELISA agreed with the results obtained from the standard MAT.

Cite This Article

APA
Yan W, Saleem MH, McDonough P, McDonough SP, Divers TJ, Chang YF. (2013). Development of an enzyme-linked immunosorbent assay using a recombinant LigA fragment comprising repeat domains 4 to 7.5 as an antigen for diagnosis of equine leptospirosis. Clin Vaccine Immunol, 20(8), 1143-1149. https://doi.org/10.1128/CVI.00245-13

Publication

Clinical and vaccine immunology : CVI
ISSN: 1556-679X
NlmUniqueID: 101252125
Country: United States
Language: English
Volume: 20
Issue: 8
Pages: 1143-1149

Researcher Affiliations

Yan, Weiwei
  • Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Saleem, Muhammad Hassan
    McDonough, Patrick
      McDonough, Sean P
        Divers, Thomas J
          Chang, Yung-Fu

            MeSH Terms

            • Animals
            • Antibodies, Bacterial / blood
            • Antigens, Bacterial / genetics
            • Clinical Laboratory Techniques / methods
            • Enzyme-Linked Immunosorbent Assay / methods
            • Horse Diseases / diagnosis
            • Horses
            • Immunoblotting / methods
            • Leptospira / genetics
            • Leptospira / immunology
            • Leptospirosis / diagnosis
            • Leptospirosis / veterinary
            • Recombinant Proteins / genetics
            • Sensitivity and Specificity
            • Veterinary Medicine / methods

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