title: 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.
authors.affiliation: Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Yan
authors.forename: Muhammad Hassan
authors.initials: MH
authors.lastname: Saleem
authors.forename: Patrick
authors.initials: P
authors.lastname: McDonough
authors.forename: Sean P
authors.initials: SP
authors.lastname: McDonough
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Divers
authors.forename: Yung-Fu
authors.initials: YF
authors.lastname: Chang
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006734:Horse Diseases / Q000175:diagnosis*; D006736:Horses; D015151:Immunoblotting / Q000379:methods; D007919:Leptospira / Q000235:genetics / Q000276:immunology*; D007922:Leptospirosis / Q000175:diagnosis / Q000662:veterinary*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; C487005:LigA-m protein, Leptospira interrogans; D011994:Recombinant Proteins