FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
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.
Get Full Text
Publication Date: 2013-05-29 PubMed ID: 23720368PubMed Central: PMC3754523DOI: 10.1128/CVI.00245-13Google 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
  • 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

            References

            This article includes 41 references
            1. Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen.. Nat. Rev. Microbiol. 7:736–747.
              pmc: PMC3384523pubmed: 19756012
            2. Palaniappan RU, Ramanujam S, Chang YF. Leptospirosis: pathogenesis, immunity, and diagnosis.. Curr. Opin. Infect. Dis. 20:284–292.
              pubmed: 17471039
            3. Divers TJ, Byars TD, Shin SJ. Renal dysfunction associated with infection of Leptospira interrogans in a horse.. J. Am. Vet. Med. Assoc. 201:1391–1392.
              pubmed: 1429185
            4. Faisal SM, McDonough SP, Chang YF. Leptospira: invasion, pathogenesis and persistence, p 143–172 In Embers ME. (ed), The pathogenic spirochetes: strategies for evasion of host immunity and persistence.. Springer Science New York, NY.
            5. Broux B, Torfs S, Wegge B, Deprez P, van Loon G. Acute respiratory failure caused by Leptospira spp. in 5 foals.. J. Vet. Intern. Med. 26:684–687.
              pubmed: 22404543
            6. Cerri D, Ebani VV, Fratini F, Pinzauti P, Andreani E. Epidemiology of leptospirosis: observations on serological data obtained by a “diagnostic laboratory for leptospirosis” from 1995 to 2001.. New Microbiol. 26:383–389.
              pubmed: 14596349
            7. Levett PN. Leptospirosis.. Clin. Microbiol. Rev. 14:296–326.
              pmc: PMC88975pubmed: 11292640
            8. Toyokawa T, Ohnishi M, Koizumi N. Diagnosis of acute leptospirosis.. Expert Rev. Anti Infect. Ther. 9:111–121.
              pubmed: 21171882
            9. Woodward MJ, Swallow C, Kitching A, Dalley C, Sayers AR. Leptospira Hardjo serodiagnosis: a comparison of MAT, ELISA and Immunocomb.. Vet. Rec. 141:603–604.
              pubmed: 9429279
            10. Bomfim MR, Ko A, Koury MC. Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.. Vet. Microbiol. 109:89–94.
              pubmed: 15950404
            11. Chalayon P, Chanket P, Boonchawalit T, Chattanadee S, Srimanote P, Kalambaheti T. Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.. Trans. R. Soc. Trop. Med. Hyg. 105:289–297.
              pubmed: 21353274
            12. Flannery B, Costa D, Carvalho FP, Guerreiro H, Matsunaga J, Da Silva ED, Ferreira AG, Riley LW, Reis MG, Haake DA, Ko AI. Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis.. J. Clin. Microbiol. 39:3303–3310.
              pmc: PMC88335pubmed: 11526167
            13. Croda J, Ramos JG, Matsunaga J, Queiroz A, Homma A, Riley LW, Haake DA, Reis MG, Ko AI. Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis.. J. Clin. Microbiol. 45:1528–1534.
              pmc: PMC1865864pubmed: 17360842
            14. Hartleben CP, Leal FM, Monte LG, Hartwig DD, Seixas FK, Vasconcellos SA, Brihuega B, Dellagostin OA. Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.. Curr. Microbiol. 66:106–109.
              pubmed: 23064970
            15. Joseph S, Thomas N, Thangapandian E, Singh VP, Verma R, Srivastava SK. Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis.. J. Vet. Sci. 13:99–101.
              pmc: PMC3317464pubmed: 22437542
            16. Oliveira TR, Longhi MT, de Morais ZM, Romero EC, Blanco RM, Kirchgatter K, Vasconcellos SA, Nascimento AL. Evaluation of leptospiral recombinant antigens MPL17 and MPL21 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays.. Clin. Vaccine Immunol. 15:1715–1722.
              pmc: PMC2583518pubmed: 18799647
            17. Sankar S, Harshan HM, Somarajan SR, Srivastava SK. Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.. Res. Vet. Sci. 88:375–378.
              pubmed: 20022618
            18. Srimanote P, Wongdeethai N, Jieanampunkul P, Samonkiert S, Leepiyasakulchai C, Kalambaheti T, Prachayasittikul V. Recombinant ligA for leptospirosis diagnosis and ligA among the Leptospira spp. clinical isolates.. J. Microbiol. Methods 72:73–81.
              pubmed: 18079011
            19. Nabity SA, Ribeiro GS, Aquino CL, Takahashi D, Damiao AO, Goncalves AH, Miranda-Filho DB, Greenwald R, Esfandiari J, Lyashchenko KP, Reis MG, Medeiros MA, Ko AI. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis.. PLoS Negl. Trop. Dis. 6:e1878.
              doi: 10.1371/journal.pntd.0001878pmc: PMC3486890pubmed: 23133686google scholar: lookup
            20. Lin YP, McDonough SP, Sharma Y, Chang YF. Leptospira immunoglobulin-like protein B (LigB) binding to the C-terminal fibrinogen αC domain inhibits fibrin clot formation, platelet adhesion and aggregation.. Mol. Microbiol. 79:1063–1076.
              pubmed: 21219469
            21. Lin YP, Lee DW, McDonough SP, Nicholson LK, Sharma Y, Chang YF. Repeated domains of Leptospira immunoglobulin-like proteins interact with elastin and tropoelastin.. J. Biol. Chem. 284:19380–19391.
              pmc: PMC2740563pubmed: 19473986
            22. Lin YP, Greenwood A, Nicholson LK, Sharma Y, McDonough SP, Chang YF. Fibronectin binds to and induces conformational change in a disordered region of leptospiral immunoglobulin-like protein B.. J. Biol. Chem. 284:23547–23557.
              pmc: PMC2749129pubmed: 19581300
            23. Lin YP, Raman R, Sharma Y, Chang YF. Calcium binds to leptospiral immunoglobulin-like protein, LigB, and modulates fibronectin binding.. J. Biol. Chem. 283:25140–25149.
              pubmed: 18625711
            24. Lin YP, Chang YF. A domain of the Leptospira LigB contributes to high affinity binding of fibronectin.. Biochem. Biophys. Res. Commun. 362:443–448.
              pubmed: 17707344
            25. Choy HA, Kelley MM, Chen TL, Moller AK, Matsunaga J, Haake DA. Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen.. Infect. Immun. 75:2441–2450.
              pmc: PMC1865782pubmed: 17296754
            26. Palaniappan RU, Chang YF, Hassan F, McDonough SP, Pough M, Barr SC, Simpson KW, Mohammed HO, Shin S, McDonough P, Zuerner RL, Qu J, Roe B. Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA.. J. Med. Microbiol. 53:975–984.
              pubmed: 15358819
            27. Palaniappan RU, Chang YF, Jusuf SS, Artiushin S, Timoney JF, McDonough SP, Barr SC, Divers TJ, Simpson KW, McDonough PL, Mohammed HO. Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans.. Infect. Immun. 70:5924–5930.
              pmc: PMC130282pubmed: 12379666
            28. Palaniappan RU, McDonough SP, Divers TJ, Chen CS, Pan MJ, Matsumoto M, Chang YF. Immunoprotection of recombinant leptospiral immunoglobulin-like protein A against Leptospira interrogans serovar Pomona infection.. Infect. Immun. 74:1745–1750.
              pmc: PMC1418682pubmed: 16495547
            29. Cole JR Jr, Sulzer CR, Pursell AR. Improved microtechnique for the leptospiral microscopic agglutination test.. Appl. Microbiol. 25:976–980.
              pmc: PMC380950pubmed: 4736794
            30. Zhu D, Saul AJ, Miles AP. A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli.. J. Immunol. Methods 306:40–50.
              pubmed: 16137696
            31. Yan W, Faisal SM, Divers T, McDonough SP, Akey B, Chang YF. Experimental Leptospira interrogans serovar Kennewicki infection of horses.. J. Vet. Intern. Med. 24:912–917.
              pubmed: 20649749
            32. Faine S, Adher B, Bloin C, Perolat P. Leptospira and leptospirosis, 2nd ed. MedSci Melbourne, Australia.
            33. Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NP, Cooper B, Peacock SJ. Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.. Clin. Infect. Dis. 55:322–331.
              pmc: PMC3393707pubmed: 22523263
            34. Matsunaga J, Barocchi MA, Croda J, Young TA, Sanchez Y, Siqueira I, Bolin CA, Reis MG, Riley LW, Haake DA, Ko AI. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.. Mol. Microbiol. 49:929–945.
              pmc: PMC1237129pubmed: 12890019
            35. Burke E, Barik S. Specific detection of native multi-subunit proteins by slot-blot assay.. Biotechniques 25:574–576, 578, 582.
              pubmed: 9793635
            36. Chapman AJ, Adler B, Faine S. Antigens recognised by the human immune response to infection with Leptospira interrogans serovar Hardjo.. J. Med. Microbiol. 25:269–278.
              pubmed: 3282073
            37. Chapman AJ, Everard CO, Faine S, Adler B. Antigens recognized by the human immune response to severe leptospirosis in Barbados.. Epidemiol. Infect. 107:143–155.
              pmc: PMC2272025pubmed: 1879481
            38. Guerreiro H, Croda J, Flannery B, Mazel M, Matsunaga J, Galvao Reis M, Levett PN, Ko AI, Haake DA. Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans.. Infect. Immun. 69:4958–4968.
              pmc: PMC98588pubmed: 11447174
            39. Engstrom SM, Shoop E, Johnson RC. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease.. J. Clin. Microbiol. 33:419–427.
              pmc: PMC227960pubmed: 7714202
            40. Magnarelli LA, Ijdo JW, Padula SJ, Flavell RA, Fikrig E. Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens.. J. Clin. Microbiol. 38:1735–1739.
              pmc: PMC86574pubmed: 10790090
            41. Schmidt BL, Edjlalipour M, Luger A. Comparative evaluation of nine different enzyme-linked immunosorbent assays for determination of antibodies against Treponema pallidum in patients with primary syphilis.. J. Clin. Microbiol. 38:1279–1282.
              pmc: PMC88607pubmed: 10699042

            Citations

            This article has been cited 9 times.
            1. Cardoso TL, Wozeak DR, Pereira IL, da Silva Ribeiro LD, Rodrigues RO, Hartwig DD. Advances in ELISA-based detection of equine leptospirosis. Braz J Microbiol 2026 Feb 9;57(1):52.
              doi: 10.1007/s42770-025-01854-zpubmed: 41663676google scholar: lookup
            2. Sueasuay J, Boonwong C, Saisuwan R, Kassaket N, Prachongsai I, Tantibhedhyangkul W, Wongprompitak P, Suputtamongkol Y, Ekpo P, Inthasin N. Evaluation of the conserved subunit of the pathogenic Leptospira FlaB protein-based immunochromatographic test for the diagnosis of acute leptospirosis. Microbiol Spectr 2026 Jan 6;14(1):e0030725.
              doi: 10.1128/spectrum.00307-25pubmed: 41277860google scholar: lookup
            3. Behera SK, Sabarinath T, Ganesh B, Mishra PKK, Niloofa R, Senthilkumar K, Verma MR, Hota A, Chandrasekar S, Deneke Y, Kumar A, Nagarajan M, Das D, Khatua S, Sahu R, Ali SA. Diagnosis of Human Leptospirosis: Comparison of Microscopic Agglutination Test with Recombinant LigA/B Antigen-Based In-House IgM Dot ELISA Dipstick Test and Latex Agglutination Test Using Bayesian Latent Class Model and MAT as Gold Standard. Diagnostics (Basel) 2022 Jun 13;12(6).
              doi: 10.3390/diagnostics12061455pubmed: 35741265google scholar: lookup
            4. Haake DA, Matsunaga J. Leptospiral Immunoglobulin-Like Domain Proteins: Roles in Virulence and Immunity. Front Immunol 2020;11:579907.
              doi: 10.3389/fimmu.2020.579907pubmed: 33488581google scholar: lookup
            5. Xu K, Chi S, Chen B, Chen L, Zheng D. The Distribution of Syphilis Among Inpatients in Wenzhou, China: A Hospital Based Study. Jundishapur J Microbiol 2016 Aug;9(8):e27825.
              doi: 10.5812/jjm.27825pubmed: 27800128google scholar: lookup
            6. Nagalingam M, Thirumalesh SR, Kalleshamurthy T, Niharika N, Balamurugan V, Shome R, Sengupta PP, Shome BR, Prabhudas K, Rahman H. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis. Trop Anim Health Prod 2015 Oct;47(7):1329-35.
              doi: 10.1007/s11250-015-0867-7pubmed: 26065562google scholar: lookup
            7. Ye C, Yan W, Xiang H, He H, Yang M, Ijaz M, Useh N, Hsieh CL, McDonough PL, McDonough SP, Mohamed H, Yang Z, Chang YF. Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs. PLoS One 2014;9(12):e111367.
              doi: 10.1371/journal.pone.0111367pubmed: 25526513google scholar: lookup
            8. Fabres-Klein MH, Aguilar AP, Silva MP, Silva DM, Ribon AO. Moving towards the immunodiagnosis of staphylococcal intramammary infections. Eur J Clin Microbiol Infect Dis 2014 Dec;33(12):2095-104.
              doi: 10.1007/s10096-014-2181-0pubmed: 24947175google scholar: lookup
            9. Ye C, Yan W, McDonough PL, McDonough SP, Mohamed H, Divers TJ, Chang YF, Yang Z. Serodiagnosis of equine leptospirosis by enzyme-linked immunosorbent assay using four recombinant protein markers. Clin Vaccine Immunol 2014 Apr;21(4):478-83.
              doi: 10.1128/CVI.00649-13pubmed: 24451330google scholar: lookup
            Subscribe to our newsletter
            Join 99,928 horse owners like you who receive our email updates on horse health and nutrition.