- Original Article
- Open Access
Development of competitive ELISA for neosporosis by employing immunoproteomics
Clinical Proteomics volume 1, Article number: 235 ()
In this study, proteomics was used to explore the antigenic proteins that are involved in cross-reactivity during serodiagnosis between Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Competitive enzyme-linked immunosorbent assay (C-ELISA) developed by proteomics shed a new light on the infection of N. caninum. Cross-reactivity of antigenic proteins between N. caninum and T. gondii tachyzoites was explored by using the conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (1-DE) and two-dimensional gel electrophoresis (2-DE) immunoblot. The proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein expression patterns in the immunoblot profiles of N. caninum were similar to bovine, chicken, and rabbit anti-N. caninum serum, but they were not similar to rabbit anti-T. gondii serum. Band at 79 kDa, HSP70, and actin on immunoblot profiles reacted, in general, with bovine, chicken, and rabbit anti-N. caninum serum and also with rabbit anti-T. gondii serum, respectively. Whereas the band at 144 kDa, and NCDG-1 were detected on bovine, chicken, and rabbit anti-N. caninum immunoblot profiles, they were not observed on rabbit anti-T. gondii immunoblot profile. These specific antigenic proteins were recorded as species-specific proteins of N. caninum against T. gondii. Based on the proteome analysis, C-ELISA was developed to screen the cattle infected with N. caninum by using N. caninum tachyzoite lysate as a coating antigen and chicken anti-N. caninum immunoglobulin (Ig)Y as a competitor. C-ELISA was able to detect the antibody of N. caninum without cross-reactivity with T. gondii. Furthermore, it achieved a fine diagnostic performance in the cases of 162 bovine sera.
Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988; 192:1269–1285.
Bjerkås I, Presthus J. Immuno-histochemical and ultrastructural characteristics of a cyst-forming sporozoon associated with encephalo-myelitis and myositis in dogs. Apmis 1988; 96:445–454.
Bjerkås I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd 1984; 70:271–274.
Dubey JP. Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 2003;41:1–16.
Jenkins M, Baszler T, Björkman C, Schares G, Williams D. Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion. Int J Parasitol 2002;32:631–636.
Björkman C, Uggla A. Serological diagnosis of Neospora caninum infection. Int J Parasitol 1999;29:1497–1507.
Dubey JP, Lindsay DS, Adams DS, Gay JM, Baszler TV, Blagburn BL, Thulliez P. Serologic responses of cattle and other animals infected with Neospora caninum. Am J Vet Res 1996; 57:329–336.
Wouda W, Brinkhof J, van Maanen C, de Gee AL, Moen AR. Serodiagnosis of neosporosis in individual cows and dairy herds: A comparative study of three enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol 1998; 5:711–716.
Atkinson R, Harper PA, Reichel MP, Ellis JT. Progress in the serodiagnosis of Neospora caninum infections of cattle. Parasitol Today 2000;16:110–114.
Howe DK, Tang K, Conrad PA, Sverlow K, Dubey JP, Sibley LD. Sensitive and specific identification of Neospora caninum infection of cattle based on detection of serum antibodies to recombinant Ncp29. Clin Diagn Lab Immunol 2002;9:611–615.
Ahn HJ, Kim S, Kim DY, Nam HW. ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera. Korean J Parasitol 2003;41:175–177.
Heckeroth A, Tenter A, Hemphill A, Innenes E, Buxton D. Evaluation of cross-reactivity between tachyzoites of Neospora caninum and Toxoplasma gondii by two-dimensional gel electrophoresis. In: Hemphill A, Gottstein B, ed. A European perspective on Neospora caninum, Vol. 30. Int J Parasitol, 2000:877–924.
Lee EG, Kim JH, Shin YS, Shin GW, Suh MD, Kim DY, Kim YH, Kim GS, Jung TS. Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics. Proteomics 2003;3:2339–2350.
Kim JH, Sohn HJ, Hwang WS, Hwang EK, Jean YH, Yamane I, Kim DY. In vitro isolation and characterization of bovine Neospora caninum in Korea. Vet Parasitol 2000;90:147–154.
Baszler TV, Adams S, Vander-Schalie J, Mathison BA, Kostovic M. Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle. J Clin Microbiol 2001;39:3851–3857.
Pappas MG, Lunde MN, Hajkowski R, McMahon J. Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures. Vet Parasitol 1996;20:31–42.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.
Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:1037–1053.
Mansfield MA. Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking. Anal Biochem 1995;229:140–143.
Crowther J. Stages in ELISA. In: Crowther J, ed. ELISA; theory and practice. New Jersey: Humana press, 1995:63–98.
Osawa T, Wastling J, Maley S, Buxton D, Innes EA. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera. Vet Parasitol 1998;79:19–34.
Baszler TV, Knowles DP, Dubey JP, Gay JM, Mathison BA, McElwain TF. Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. J Clin Microbiol 1996;34:1423–1428.
Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159–174.
Barta JR, Dubey JP. Characterization of anti Neospora caninum hyperimmune rabbit serum by western blot analysis and immunoelectron microscopy. Parasitol Res 1992;78:689–694.
Bjerkås I, Jenkins MC, Dubey JP. Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis. Clin Diagn Lab Immunol 1994;1: 214–221.
Harkins D, Clements DN, Maley S, Marks J, Wright S, Esteban I, Innes EA, Buxton D. Western blot analysis of the IgG responses of ruminants infected with Neospora caninum and with Toxoplasma gondii. J Comp Pathol 1998;119:45–55.
Stenlund S, Björkman C, Holmdahl OJ, Kindahl H, Uggla A. Characterization of a Swedish bovine isolate of Neospora caninum. Parasitol Res 1997;83:214–219.
Nam HW, Kang SW, Choi WY. Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens. Korean J Parasitol 1998;36:269–275.
Tranas J, Heinzen RA, Weiss LM, McAllister MM. Serological evidence of human infection with the protozoan Neospora caninum. Clin Diagn Lab Immunol 1999;6:765–767.
Dobrowolski J, Sibley LD. The role of the cytoskeleton in host cell invasion by Toxoplasma gondii. Behring Inst Mitt 1997;99:90–96.
Augustine PC, Jenkins MC, Dubey JP. Effect of polyclonal antisera developed against dense granule-associated Neospora caninum proteins on cell invasion and development in vitro by N. caninum tachyzoites. Parasitology 1999;119: 441–445.
Dobbin CA, Smith NC, Johnson AM. Heat shock protein 70 is a potential virulence factor in murine toxoplasma infection via immuno-modulation of host NF-kappa B and nitric oxide. J Immunol 2002;169:958–965.
Hughes HP, Van Knapen F, Atkinson HJ, Balfour AH, Lee DL. A new soluble antigen preparation of Toxoplasma gondii and its use in serological diagnosis. Clin Exp Immunol 1982; 49:239–246.
Uggla A, Buxton D. Immune responses against Toxoplasma and Sarcocystis infections in ruminants: diagnosis and prospects for vaccination. Rev Sci Tech 1990;9:441–462.
Gassmann M, Thommes P, Weiser T, Hubscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. Faseb J 1990;4:2528–2532.
Hatta H, Tsuda K, Akachi S, Kim M, Yamamoto T. Productivity and some properties of egg yolk antibody (IgY) against human rotavirus compared with rabbit IgG. Biosci Biotechnol Biochem 1993;57:450–454.
Behn I, Hommel U, Erhard M, Hlinak A, Schade R, Schwarzkopf C, Staak C. Use of polyclonal avian antibodies. In: Schade R, Behn I, Erhard M, Hlinak A, Staak C. eds, Chicken egg yolk antibodies, production and application: IgY-thechnology. Heidelberg: Springer-verlag, 2001: 108–210.
Devi CM, Bai MV, Lal AV, Umashankar PR, Krishnan LK. An improved method for isolation of anti-viper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 2002;51:129–138.
Zhang WW. The use of gene-specific IgY antibodies for drug target discovery. Drug Discov Today 2003;8:364–371.
Paré J, Hietala SK, Thurmond MC. An enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora sp. infection in cattle. J Vet Diagn Invest 1995;7: 352–359.
Björkman C, Lunden A, Holmdahl J, Barber J, Trees AJ, Uggla A. Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen. Parasite Immunol 1994; 16:643–648.
Björkman C, Holmdahl OJ, Uggla A. An indirect enzyme-linked immunoassay (ELISA) for demonstration of antibodies to Neospora caninum in serum and milk of cattle. Vet Parasitol 1997;68:251–260.
Conrad PA, Sverlow K, Anderson M, Rowe J, BonDurant R, Tuter G, Breitmeyer R, Palmer C, Thurmond M, Ardans A, and et al. Detection of serum antibody responses in cattle with natural or experimental Neospora infections. J Vet Diagn Invest 1993;5:572–578.
Paré J, Thurmond MC, Hietala SK. Neospora caninum antibodies in cows during pregnancy as a predictor of congenital infection and abortion. J Parasitol 1997;83:82–87.
About this article
Cite this article
Shin, Y., Lee, E., Shin, G. et al. Development of competitive ELISA for neosporosis by employing immunoproteomics. Clin Proteom 1, 235 (2004) doi:10.1385/CP:1:3-4:235
- Neospora caninum
- Toxoplasma gondii
- competitive ELISA