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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.
AuthorsWeiwei Yan, Muhammad Hassan Saleem, Patrick McDonough, Sean P McDonough, Thomas J Divers, Yung-Fu Chang
JournalClinical and vaccine immunology : CVI (Clin Vaccine Immunol) Vol. 20 Issue 8 Pg. 1143-9 (Aug 2013) ISSN: 1556-679X [Electronic] United States
PMID23720368 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Antibodies, Bacterial
  • Antigens, Bacterial
  • LigA-m protein, Leptospira interrogans
  • Recombinant Proteins
Topics
  • Animals
  • Antibodies, Bacterial (blood)
  • Antigens, Bacterial (genetics)
  • Clinical Laboratory Techniques (methods)
  • Enzyme-Linked Immunosorbent Assay (methods)
  • Horse Diseases (diagnosis)
  • Horses
  • Immunoblotting (methods)
  • Leptospira (genetics, immunology)
  • Leptospirosis (diagnosis, veterinary)
  • Recombinant Proteins (genetics)
  • Sensitivity and Specificity
  • Veterinary Medicine (methods)

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