Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA.
OBJECTIVE
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.


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 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.


CONCLUSIONS
The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.
Blais
B W
BW
Ottawa Laboratory (Carling), Canadian Food Inspection Agency, CEF, Ottawa, Canada. bblais@inspection.gc.ca
Leggate
J
J
Bosley
J
J
Martinez-Perez
A
A
D000469:Alkaline Phosphatase / Q000032:analysis; D000907:Antibodies, Bacterial / Q000737:chemistry; D001431:Bacteriological Techniques; D001560:Benzidines / Q000378:metabolism; D002863:Chromogenic Compounds / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005516:Food Microbiology; D008070:Lipopolysaccharides / Q000737:chemistry; D008460:Meat / Q000382:microbiology; D009195:Peroxidase / Q000032:analysis; D011113:Polymyxins; D011485:Protein Binding; D014675:Vegetables / Q000382:microbiology
D000907:Antibodies, Bacterial; D001560:Benzidines; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D011113:Polymyxins; C021758:3,3',5,5'-tetramethylbenzidine; D009195:Peroxidase; D000469:Alkaline Phosphatase