Folia Microbiologica

, Volume 49, Issue 5, pp 579–586 | Cite as

Development of an indirect competitive ELISA for detection ofCampylobacter jejuni subsp.jejuni O:23 in foods

  • I. Hochel
  • D. Viochna
  • J. Škvor
  • M. Musil
Article

Abstract

An indirect enzyme immunoassay for rapid detection ofCampylobacter jejuni subsp.jejuni O:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 µg/mL, and 8 µg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/µL, limit of quantification being 480 CFU/µL. By testing 5 chromogens.viz. 1,2-benzenediamine, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3′,5,5′-tetramethylbenzidine, bi(4,4′-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3′,5,5′-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain ofC. sputorum subsp.sputorum (21.5 %) and with G+ bacteriumMicrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according toČSN ISO 10272 and commercially available Singlepath® Campylobacter GLISA-Rapid Test.

Abbreviations

ABS

2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)

BAN

bi(4,4′-anisidine) (3,3′-dimethoxybenzidine)

BDA

1,2-benzenediamine

BSA

bovine serum albumin

DAB

3-dimethylaminobenzoic acid

HRP

horseradish peroxidase

MBH

3-methyl-2-benzothiazolinone hydrazone

PBS

phosphate buffered saline

TMB

3,3′,5,5′-tetramethylbenzidine

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Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic 2004

Authors and Affiliations

  • I. Hochel
    • 1
  • D. Viochna
    • 1
  • J. Škvor
    • 2
  • M. Musil
    • 1
  1. 1.Department of Biochemistry and MicrobiologyInstitute of Chemical TechnologyPrague 6Czechia
  2. 2.Department of Anthropology and Human Genetics, Faculty of ScienceCharles UniversityPrague 2Czechia

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