Filtration capture immunoassay for bacteria: optimization and potential for urinalysis.

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 microm 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 electrochemical detection produced a 25-min assay with a detection limit of 5 x 10(3) E. coli O157:H7 per ml using a filtration volume of 100 microl (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.     

Analysis of herbicides: demonstration of the utility of enzyme immunoassay verification by HPLC

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.     

Analysis of herbicides: demonstration of the utility of enzyme immunoassay verification by HPLC

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Chemostat selection of a bacterial community able to degrade s-triazinic compounds: continuous simazine biodegradation in a multi-stage packed bed biofilm reactor

Using a successive transfer method on mineral salt medium containing simazine, a microbial community enriched with microorganisms able to grow on simazine was obtained. Afterwards, using a continuous enrichment culture procedure, a bacterial community able to degrade simazine from an herbicide formulation was isolated from a chemostat. The continuous selector, fed with a mineral salt medium containing simazine and adjuvants present in the commercial herbicide formulation, was maintained in operation for 42 days. Following the lapse of this time, the cell count increased from 5 x 10(5) to 3 x 10(8) CFU mL(-1), and the simazine removal efficiency reached 96%. The chemostat's bacterial diversity was periodically evaluated by extracting the culture's bacterial DNA, amplifying their 16S rDNA fragments and analyzing them by thermal gradient gel electrophoresis. Finally, a stable bacterial consortium able to degrade simazine was selected. By PCR amplification, sequencing of bacterial 16S rDNA amplicons, and comparison with known sequences of 16S rDNA from the NCBI GenBank, eight bacterial strains were identified. The genera, Ochrobactrum, Mycobacterium, Cellulomonas, Arthrobacter, Microbacterium, Rhizobium and Pseudomonas have been reported as common degraders of triazinic herbicides. On the contrary, we were unable to find reports about the ability of the genus Pseudonocardia to degrade triazinic compounds. The selected bacterial community was attached to a porous support in a concurrently aerated four-stage packed-bed reactor fed with the herbicide. Highest overall simazine removal efficiencies eta (SZ) were obtained at overall dilution rates D below 0.284 h(-1). However, the multistage packed bed reactor could be operated at dilution rates as high as D = 3.58 h(-1) with overall simazine removal volumetric rates R (v,SZ) = 19.6 mg L(-1) h(-1), and overall simazine removal specific rates R (X,SZ) = 13.48 mg (mg cell protein)(-1) h(-1). Finally, the consortium's ability to degrade 2-chloro-4,6-diamino-1,3,5-triazine (CAAT), cyanuric acid and the herbicide atrazine, pure or mixed with simazine, was evaluated in fed batch processes.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains

AIMS: To improve the sensitivity of a monoclonal antibody (MAb 2F3) based enteropathogenic Escherichia coli (EPEC)/enterohaemorrhagic E. coli (EHEC) serogroup O26-specific sandwich ELISA (sELISA), using a capture/enrichment format of the assay. METHODS AND RESULTS: The sELISA utilized an EPEC/EHEC O26-specific MAb 2F3 as the capture reagent and an E. coli serogroup O26 lipopolysaccharide-specific polyclonal antibody in the development stage. Wells containing faeces test samples from bovine enteritis cases and agar colony sweep cultures from human diarrhoea cases, after a 2-h capture stage, were washed and enrichment of the captured cells was encouraged by addition of tryptone soya broth. After overnight incubation, the contents of each well were transferred to sterile wells and the sELISA completed. Any sELISA positive samples were then subcultured onto blood agar to recover and further characterize the positive cultures. The assay had a sensitivity of 10(3) CFU ml(-1). ELISA positive samples consisted of 21 (4.8%) of the 442 bovine and 19 (3.7%) of the 519 human samples tested, and ELISA positive EPEC/EHEC O26 strains were isolated from 11 and three of these samples respectively. CONCLUSION: The capture/enrichment method improved the sensitivity of a MAb-based sELISA for the detection of EPEC/EHEC O26 strains, and also contributed to an improved isolation rate of the organism from field samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a specific MAb in a capture/enrichment format of the sELISA, provides a prospectively suitable screening method for the detection of pathogenic bacteria from mixed culture samples.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).     

An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis

A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.     

Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

Mitochondrial and cytosolic hexokinase from rat brain: one and the same enzyme?

Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.     

Evaluation of progesterone content in saliva using magnetic particle-based immuno supported liquid membrane assay (m-ISLMA)

Progesterone in saliva was monitored using a new method called magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) in a sequential injection (SI) setup, allowing automatic sample cleanup, analyte enrichment, and detection in a single analysis unit. Progesterone (Ag) diffuses from a continuous flowing sample - the donor - into a supported organic liquid membrane (SLM), based on analyte partitioning (solubility) between the aqueous donor and the organic phase. The Ag is re-extracted from the SLM into a second stagnant aqueous acceptor, containing antibodies (Ab) immobilized on magnetic beads, held at the bottom of the acceptor by a magnet. Due to the formation of strong Ag-Ab-bead complexes and a large excess of Ab-beads, the Ag is accumulated and selectively enriched in the acceptor. The extracted progesterone was quantified by injecting into the acceptor a horseradish peroxidase (HRP) labeled analyte tracer, the substrate (luminol, H(2)O(2), and p-iodophenol), and finally detection of the generated chemiluminescence by a photomultiplier tube. After optimization of experimental parameters (e.g., sample flow rate, extraction time, type of organic solvent and antibody-bead concentration in the acceptor), a detection limit of 8.50+/-0.17 fgL(-1) and a dynamic range between 35 fgL(-1) and 10 pgL(-1) was reached. The progesterone level of saliva for three subjects (women in different period of ovarian cycle) was investigated, and the corresponding progesterone concentrations detected with m-ISLMA coincided well with the expected values.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

On true and apparent Michaelis constants in enzymology. III. Is it linear dependence between apparent michaelis constant and limiting rate and is it possible to determine the substrate constant value using this dependence?

The Slater-Bonner method which is used for graphic determination of substrate constant (Ks) by linear dependence of apparent Michaelis constant (Km(app)) on the limiting rate (V(app)) of enzyme-catalysed reactions with activator participation has been critically analysed. It has been shown that although it is possible to record the mechanisms of such reactions as a scheme similar to Michaelis-Menten model which allow to find correlation Km(app) and V(app) as equation Km(app) = Ks + V(app)/k1[E]0 ([E]0 is a total enzyme concentration, k1 is a rate constant of enzyme-substrate complex formation from free enzyme and substrate) in order to calculate Ks and individual rate constants (k1, k(-1)), but this approach for investigation of all reactions with activator participation ought not to be used. The above equation is not obeyed in general, it may be true for some mechanisms only or under certain ratios of kinetic parameters of enzyme-catalysed reactions.     

鼠冠状病毒核衣壳蛋白的抗体制备与抗原决定簇分析

核衣壳(nucleocapsid,N)蛋白有稳定病毒基因组、调控病毒复制及细胞状态的特殊作用。鼠肝炎病毒(murine hepatitis virus,MHV)为乙型冠状病毒属的原型病毒,是研究冠状病毒N蛋白功能的经典模型。本研究用去污剂处理鼠冠状病毒粒子暴露N蛋白,另用原核表达纯化的重组N蛋白分别免疫小鼠,制备多克隆及单克隆抗体。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹分析结果显示两类抗体均具有高灵敏度和特异度,与甲型冠状病毒猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)的N蛋白无交叉反应。原核表达缺失突变的N蛋白分析结果显示,多克隆抗体与单克隆抗体2E6识别的鼠冠状病毒N蛋白抗原决定簇完全一致,位于N端结构域(N-terminal domain,NTD)C端与SR之间的58个氨基酸残基内。此外,基于单克隆抗体2E6的ELISA及免疫荧光法能检测到感染细胞中和培养上清液中的N蛋白组分,且其含量与病毒复制的滴度一致。这些结果表明,鼠冠状病毒复制过程中粒子与细胞中的N蛋白可能维持相似的结构,使NTD与SR之间的部分氨基酸残基一直暴露在表面,从而形成了优势抗原决定簇。     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.