Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7 (original) (raw)
Related papers
Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7
Journal of Immunological Methods, 1998
A new approach for rapid assay of bacteria in liquid samples is described. Cells were labeled by incubation with an enzyme-antibody conjugate and captured by filtration of the samplerconjugate mixture through a 0.2 mm filter. The enzyme-labeled cells were detected by placing the filter on the surface of an electrode, incubating with enzyme substrate, and measuring the current produced by oxidation of the electroactive enzyme product. Assay time was 25 min and a detection limit of ; 5000 cellsrml was obtained for E. coli O157:H7. Background current due to non-specific binding of conjugate to the filter was the primary factor controlling the detection limit, and fewer than 50 cells could be detected when Ž. very small sample volumes 10 ml were used to minimize background current. q 1998 Elsevier Science B.V.
Highly sensitive flow-injection immunoassay system for rapid detection of bacteria
Analytica Chimica Acta, 1999
A flow-injection amperometric immunofiltration assay system for the rapid detection of total Escherichia coli and Salmonella was developed. The system is based on the use of disposable porous nylon membranes which act as a support for the immobilization of anti-E. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane and an amperometric sensor. Parameters affecting the performance of the immunofiltration assay system such as membrane pore size, non-specific binding, conjugate concentration and sample volume were studied and optimized. A sandwich scheme of immunoassay was employed and the immunofiltration system was able to specifically and directly detect 50 cells/ml of total E. coli or 50 cells/ml of Salmonella with an overall analysis time of 35 min. This immunosensor can be easily adapted for the assay of other microorganisms and may be a basis for a new class of highly sensitive and automated bioanalytical devices for the rapid quantitative detection of bacteria.
Filtration capture immunoassay for bacteria: optimization and potential for urinalysis
Journal of Microbiological Methods, 1999
A novel assay utilizing immuno-labeling, filtration, and electrochemistry for the rapid detection of bacteria has been optimized for the detection of Escherichia coli O157:H7. Bacteria were specifically labeled with alkaline phosphatase conjugated polyclonal antibodies and captured on a polycarbonate track-etched membrane filter (0.2 mm pore size). The filter was then placed directly against a glassy carbon electrode, incubated with enzyme substrate, and the product detected by square wave voltammetry. The high speed and capture efficiency of membrane filtration and inherent sensitivity of 3 electrochemical detection produced a 25-min assay with a detection limit of 5 3 10 E. coli O157:H7 per ml using a filtration volume of 100 ml (i.e. 500 cells filtered). The labeling, filtration, and electrochemical steps were optimized, and the assay performance using electrochemical and colorimetric detection methods was compared. The assay was used to detect E. coli O157:H7 that was spiked into filter-sterilized urine at clinically relevant concentrations.
Clinical Diagnostic Laboratory Immunology, 1998
E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocki...
Lateral flow assay strip for detection of Escherichia coli O157:H7
2015
The use of polyclonal antibody (IgG) has recently been applied to the detection of bacteria. We developed a lateral flow assay (LFA) strip using a specific IgG in combination with colloidal gold on a nitrocellulose membrane. A conjugate, gold-anti Escherichia coli (E. coli) O157:H7 IgG was developed in this study for the detection of E. coli O157:H7 in food. The 40 nm in size of colloidal gold nanoparticles was used to conjugate the anti-E. coli O157:H7 IgG. The optimal concentration, 12.0 µg/ml of the anti-E. coli O157:H7 IgG was determined by standard curve generated in titration method. The serially diluted of E. coli O157:H7 was detected and clearly visualized on the LFA strip as low as 10 6 CFU/ml (result not shown). The IgG raised in rabbit have shown specific binding capacity against E. coli O157:H7. No other genus of bacteria, including Salmonella typhimurium, Listeria monocytogenes and Campylobacter jejuni reacted to the IgG. The LFA strip could also detect E. coli O157:H7 in different food samples matrices after 18 h-enrichment and this result were in accordance with the results of the polymerase chain reaction (PCR) and colony count.
Development of an integrated method of concentration and immunodetection of bacteria
Analytical and bioanalytical chemistry, 2018
The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase...
Clinical Chemistry, 2006
Microbiology, Addenbrookes Hospital, Cambridge, UK, for providing the control clinical E. coli isolates. References 1. Buchanan RL, Doyle MP. Foodborne disease significance of Escherichia coli O157:H7 and other enterohemorrhagic E-coli. Food Technol 1997;51:69 -76. 2. Besser RE, Griffin PM, Slutsker L. Escherichia coli O157:H7 gastroenteritis and the hemolytic uremic syndrome: an emerging infectious disease. Annu Rev Med 1999;50:355-67. 3. Deisingh AK, Thompson M. Sequences of E. coli O157:H7 detected by a PCR-acoustic wave sensor combination. Analyst 2001;126:2153-8. 4. Brewster JD, Mazenko RS. Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7. J Immunol Methods 1998;211:1-8. 5. Crawford CG, Wijey C, Fratamico P, Tu SI, Brewster J. Immunomagneticelectrochemiluminescent detection of E.coli O157:H7 in ground beef. J Rapid Methods Autom Microbiol 2000;8:249 -64. 6. Song JM, Vo-Dinh T. Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzymelinked immunosorbent assay. Anal Chim Acta 2004;507:115-21. 7. Davis KC, Nakatsu CH, Turco R, Weagant SD, Bhunia AK. Analysis of environmental Escherichia coli isolates for virulence genes using the Taq-ManPCR system. J Appl Microbiol 2003;95:612-20. 8. Bellin T, Pulz M, Matussek A, Hempen HG, Gunzer F. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
2003
Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluoresceintagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 mg antibody/1 million ...
Optimizations needed for lateral flow assay for rapid detection of pathogenic E. coli
TURKISH JOURNAL OF BIOLOGY
Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 10 5 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.