Development of an indirect competitive ELISA for detection of Campylobacter jejuni subsp.jejuni O:23 in foods.
An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 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 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. 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 of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus 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 to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.
Hochel
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I
Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Prague 6, Czechia.
Viochna
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D
Skvor
J
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Musil
M
M
D000818:Animals; D000907:Antibodies, Bacterial; D002169:Campylobacter Infections / Q000382:microbiology; D016123:Campylobacter jejuni / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D002645:Chickens; D003429:Cross Reactions; D018153:Czech Republic; D004751:Enteritis / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005516:Food Microbiology; D006801:Humans; D011817:Rabbits; D012680:Sensitivity and Specificity
D000907:Antibodies, Bacterial