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Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin‐based ELISA
Author(s) -
Blais B.W.,
Leggate J.,
Bosley J.,
MartinezPerez A.
Publication year - 2004
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2004.01616.x
Subject(s) - chromogenic , polymyxin , escherichia coli , polymyxin b , chemistry , peroxidase , chromatography , substrate (aquarium) , enzyme , microbiology and biotechnology , biochemistry , biology , ecology , gene , antibiotics
Aims: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin‐based enzyme‐linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Methods and Results: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high‐affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti‐ E. coli O157 antibody‐enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red ® and QuantaBlu TM produced only slight improvement in the performance characteristics of the polymyxin‐ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos ® compared with the chromogenic substrate p ‐nitrophenylphosphate. Conclusions: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. Significance and Impact of the Study: The polymyxin‐ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.