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. 1991 Jul;55(3):239–245.

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

M J Champagne 1, R Higgins 1, J M Fairbrother 1, D Dubreuil 1
PMCID: PMC1263458  PMID: 1889035

Abstract

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler B., Chappel R. J., Faine S. The sensitivities of different immunoassays for detecting leptospiral antigen. Zentralbl Bakteriol Mikrobiol Hyg A. 1982 Jul;252(3):405–413. [PubMed] [Google Scholar]
  2. Bolin C. A., Zuerner R. L., Trueba G. Comparison of three techniques to detect Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine. Am J Vet Res. 1989 Jul;50(7):1001–1003. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Chapman A. J., Adler B., Faine S. Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo. J Med Microbiol. 1988 Apr;25(4):269–278. doi: 10.1099/00222615-25-4-269. [DOI] [PubMed] [Google Scholar]
  5. Chappel R. J., Adler B., Ballard S. A., Faine S., Jones R. T., Millar B. D., Swainger J. A. Enzymatic radioimmunoassay for detecting Leptospira interrogans serovar pomona in the urine of experimentally-infected pigs. Vet Microbiol. 1985 Apr;10(3):279–286. doi: 10.1016/0378-1135(85)90053-7. [DOI] [PubMed] [Google Scholar]
  6. Cinco M., Banfi E., Panfili E. Leptospiral lipopolysaccharide presence in the outer envelope: electrophoretic evidence and immunological specificity. Zentralbl Bakteriol Mikrobiol Hyg A. 1988 Nov;269(3):277–283. doi: 10.1016/s0176-6724(88)80171-8. [DOI] [PubMed] [Google Scholar]
  7. Cole J. R., Jr, Ellinghausen H. C., Rubin H. L. Laboratory diagnosis of leptospirosis of domestic animals. Proc Annu Meet U S Anim Health Assoc. 1979;(83):189–195. [PubMed] [Google Scholar]
  8. Ellis W. A., Michna S. W. Bovine leptospirosis: demonstration of leptospires of the Hebdomadis serogroup in aborted fetuses and a premature calf. Vet Rec. 1976 Nov 27;99(22):430–432. doi: 10.1136/vr.99.22.430. [DOI] [PubMed] [Google Scholar]
  9. Ellis W. A., O'Brien J. J., Cassells J. Role of cattle in the maintenance of Leptospira interrogans serotype hardjo infection in Northern Ireland. Vet Rec. 1981 Jun 27;108(26):555–557. doi: 10.1136/vr.108.26.555. [DOI] [PubMed] [Google Scholar]
  10. Fairbrother J. M. Effects of products of autolysis of tissue and urine on the viability, morphology, and antigenicity of Leptospira interrogans serovar pomona. J Clin Microbiol. 1985 Feb;21(2):189–194. doi: 10.1128/jcm.21.2.189-194.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fairbrother J. M. Serological interrelationship of Leptospira serovar and genus-specific antigens by enzyme-linked immunosorbent assay. J Clin Microbiol. 1984 Dec;20(6):1089–1093. doi: 10.1128/jcm.20.6.1089-1093.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guesdon J. L., Ternynck T., Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem. 1979 Aug;27(8):1131–1139. doi: 10.1177/27.8.90074. [DOI] [PubMed] [Google Scholar]
  13. Harboe N., Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–164. doi: 10.1111/j.1365-3083.1973.tb03798.x. [DOI] [PubMed] [Google Scholar]
  14. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kelson J. S., Adler B., Chapman A. J., Faine S. Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting. J Med Microbiol. 1988 May;26(1):47–53. doi: 10.1099/00222615-26-1-47. [DOI] [PubMed] [Google Scholar]
  16. Kendall C., Ionescu-Matiu I., Dreesman G. R. Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods. 1983 Feb 11;56(3):329–339. doi: 10.1016/s0022-1759(83)80022-2. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. LeFebvre R. B., Thiermann A. B., Foley J. Genetic and antigenic differences of serologically indistinguishable leptospires of serovar hardjo. J Clin Microbiol. 1987 Nov;25(11):2094–2097. doi: 10.1128/jcm.25.11.2094-2097.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Millar B. D., Chappel R. J., Adler B. Detection of leptospires in biological fluids using DNA hybridisation. Vet Microbiol. 1987 Oct;15(1-2):71–78. doi: 10.1016/0378-1135(87)90131-3. [DOI] [PubMed] [Google Scholar]
  20. Terpstra W. J., Ligthart G. S., Schoone G. J. Serodiagnosis of human leptospirosis by enzyme-linked-immunosorrbent-assay (ELISA). Zentralbl Bakteriol A. 1980 Aug;247(3):400–405. [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  23. Vilja P., Krohn K., Tuohimaa P. A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin. J Immunol Methods. 1985 Jan 21;76(1):73–83. doi: 10.1016/0022-1759(85)90482-x. [DOI] [PubMed] [Google Scholar]
  24. Vinh T. U., Shi M. H., Adler B., Faine S. Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo. J Gen Microbiol. 1989 Oct;135(10):2663–2673. doi: 10.1099/00221287-135-10-2663. [DOI] [PubMed] [Google Scholar]
  25. Zuerner R. L., Bolin C. A. Repetitive sequence element cloned from Leptospira interrogans serovar hardjo type hardjo-bovis provides a sensitive diagnostic probe for bovine leptospirosis. J Clin Microbiol. 1988 Dec;26(12):2495–2500. doi: 10.1128/jcm.26.12.2495-2500.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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