Modification of buffered peptone water for improved recovery of heat-injured Salmonella Typhimurium.

Rapid detection of Salmonella in foods is often limited by the high demand for the sensitivity of detection, poor physiological conditions of the target cells, and high concentration of background flora. In this study, the conditions of nonselective enrichment cultivation were modified in order to improve the quantitative detection of heat-injured Salmonella in minced meat. The effect of the modifications on the recovery was observed by means of RNA-based sandwich hybridization, which was adjusted for the quantification of Salmonella enterica 23S rRNA in crude cell extracts. The supplementation of buffered peptone water with the enzyme-controlled substrate delivery system EnBase-Flo and ferrioxamine E was shown to improve the recovery of cells in both single strain cultures and in the presence of minced meat. The presented results can be used for the development of more efficient enrichment cultivation media for faster detection of food borne Salmonella.

An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR.

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.

Sensitive Listeria spp. immunoassay based on europium(III) nanoparticulate labels using time-resolved fluorescence.

Listeria spp. are Gram-positive rod shaped bacteria found universally in the environment. Pathogenic Listeria monocytogenes is seldom harmful to healthy adults, but can cause serious disease, listeriosis, especially to pregnant women, neonates, and elderly or immunocompromised people. Conventional methods for screening Listeria in food samples are time consuming and laborious, involving the use of a range of liquid media and plate cultures. In the current study, the total analysis time was shortened by employing a sensitive Listeria assay, which was able to detect the bacteria in low concentrations. Sensitivity of the sandwich immunoassay was substantially improved by utilizing europium(III)-chelate containing latex nanoparticles as tracers. Each 107 nm nanoparticle contained approximately 31000 europium(III)-chelates which enhanced the specific activity of the label. The sensitive nanoparticulate immunoassay developed for Listeria spp. was performed in one-step and two-step formats. One-step assay was notably faster, 15 min, and simpler to execute having analytical sensitivity of 300 CFU/ml and a dynamic range of three orders of magnitude. The sensitivity, 20 CFU/ml, of the 4 h two-step assay clearly exceeded that of the one-step assay, and the dynamic range was nearly five orders of magnitude. Food and environmental samples were measured against a commercial L. monocytogenes immunoassay with good correlation. The developed sensitive assay enabled shorter sample enrichment times and, therefore, faster analysis of Listeria spp. Obviously the detection of several other bacteria can also be enhanced by applying the nanoparticle assay technology.

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood.

OBJECTIVE: To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. ANIMALS: 12 healthy dogs and 35 dogs with inflammatory processes. PROCEDURE: CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. RESULTS: Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.

Generic lanthanide fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered antibody.

Sulfa antibiotics (sulfonamides) are used in veterinary and human medicine for therapeutic and prophylactic purposes. Veterinary use can result in foodstuffs derived from animals being contaminated with residual sulfonamides. Current sulfonamide-screening methods (mainly based on bacterial growth inhibition) are slow and inaccurate, since sensitivities of bacteria to different sulfonamides vary a lot. Therefore, a rapid immunoassay that was able to detect at least 18 different sulfonamides at the MRL level (100 microg/kg) from food samples in a single reaction was developed. The assay was reproducible and adequately accurate for screening purposes. The presence of sulfonamide metabolites did not cause major assay interference. We also demonstrated reliable detection of sulfonamides from a panel of meat, milk, and serum samples with the assay.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver.

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-)(1) (n = 12) and the functional limit of detection as 3.2 ng g(-)(1) for egg (n = 6) and 11.3 ng g(-)(1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs.

Anticoccidial drugs are extensively used in the poultry industry to control the infection of the single-cell protozoa of the genus Eimeria. The most commonly used coccidiostats in poultry are the polyether ionophores such as narasin and salinomycin. This paper presents a rapid and simple method for the screening of residues of these two coccidiostatic compounds in poultry and eggs. The method is based on time-resolved fluoroimmunoassay. Sample preparation of eggs consists only of one extraction and evaporation step, and a solid phase extraction step is needed only for the muscle sample preparation. Mean recoveries were 91.0% from muscle tissue and 81.1% from eggs for both narasin and salinomycin. The performance of the assay was evaluated only for narasin because salinomycin had a cross-reactivity of 100% in the assay, and the recoveries of the compounds were not significantly different (P >0.05). The limits of detection [mean + 3 x standard deviation (SD)] of narasin were 0.56 and 0.28 microg/kg, and the limits of quantification (mean + 9 x SD) were 1.80 and 0.57 microg/kg for muscle and eggs, respectively. The coefficients of variation (CV) of the interassay precision of the method, evaluated by five replicate analyses of muscle samples spiked with 2 microg/kg of narasin and egg samples spiked with 1 microg/kg of narasin, were 4.1 and 6.4%, respectively. The CVs of intra-assay precision tests, determined by 10 replicate analyses at the above-mentioned concentration levels, were 3.8 and 4.5%, respectively.

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level.

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.

Further improvement of broad specificity hapten recognition with protein engineering.

Sulfa-antibiotics (sulfonamides) are widely used in veterinary medicine. Meat and milk from treated animals can be contaminated with sulfa residues. Current sulfonamide assays are unfit for screening of food, because they are either too laborious, insensitive or specific for a few sulfa compounds only. An immunoassay for detection of all sulfas in a single reaction would be useful for screening. Previously we have improved the broad specificity sulfa binding of antibody 27G3 with random mutagenesis and phage display. In order to improve the properties of this antibody further, mutants from the previous study were recombined and more mutations introduced. These new libraries were enriched with phage display and several different mutant antibodies were isolated. The cross-reaction profile of the best mutant was better than that of the wild-type antibody and the mutants of the previous study: it was capable of binding 10 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas 5- to 11-fold better than the mutants of the previous study.

Rapid time-resolved immunofluorometric assay for the measurement of creatine kinase in serum and whole blood samples.

A rapid and simple immunochemical method was developed for the assessment of the creatine kinase (MM) isoenzyme [CK(MM)], a protein marker linked with animal welfare and meat quality. The one-step time-resolved immunofluorometric assay produced quantitative results from serum or whole blood samples in 20 min. The analytical limit of detection (mean + 2s) for the immunoassay was 17 ng/mL (n = 6), and the functional limit of detection for the analysis of porcine whole blood samples was 426 ng/mL (n = 24). The working range of the method was linear up to 50 micro g/mL, and the within-assay precision varied between 2.1 and 10.9%. The analysis of porcine serum samples showed that the results from the immunoassay method and colorimetric CK enzyme activity determination were highly correlated (r(2) = 0.965, n = 17, p < 0.001). The practicability of the assay was demonstrated by the analysis of 300 porcine whole blood samples in a slaughterhouse environment.

Improving broad specificity hapten recognition with protein engineering.

Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.

Monitoring androstenone levels in boars by direct immunochemical analysis of serum samples.

The possibility of using blood samples for screening high levels of boar taint steroid androstenone (5α-androst-16-en-3-one) was studied both in living animals at the farm and carcasses at the slaughterhouse. The steroid was measured from boar serum and fat samples with a simple time-resolved fluoroimmunoassay. Fat samples contained androstenone in the range of 90-7500 ng/g (n=214), and 74.8% of the samples exhibited fat androstenone levels above 500 ng/g. Androstenone concentrations in blood samples were measured by direct serum assay and ranged up to 215 ng/ml (n=214). The levels of androstenone were correlated (r=0.78-0.88, P<0.001) between the serum and fat samples obtained at slaughter and serum samples taken at the farm 7-11 days before slaughter. A direct serum analysis seems to give a reliable indication of the androstenone level in fat and it can also be used in the screening of living animals.