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1.
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%.  相似文献   

2.
Staphylococcus aureus enterotoxin B (SEB) is a highly heat resistant enteric toxin with a potential as a biothreat agent. A sensitive method for the detection of staphylococcal enterotoxins is needed for food safety and food defense monitoring. The objectives of this research were to develop a competitive fluorescent immunoassay with detection of SEB below toxic levels of 1 ng/mL and to minimize sample preparation. Anti-SEB was immobilized onto carboxylated polystyrene microparticles, and SEB was labeled with fluorescein isothiocyanate (FITC). The concentrations of these reagents were optimized for the detection of SEB below 1 part per billion (1 ng/mL), and other assay conditions (sample volumes and incubation periods) were optimized. Drinking water and milk samples were spiked with 0.125-10 ng/mL SEB and were equilibrated overnight prior to analysis. The water and milk samples were directly analyzed, but heating the milk samples for 10 min at 90 degrees C improved the assay performance. SEB in samples bound with the anti-SEB linked to the latex followed by the competitive binding of SEB-FITC tracer. The excess, unbound tracer was separated by centrifugation, and the fluorescence density of the supernatant was measured. SEB was detected at levels as low as 0.125 ng/mL in drinking water and 0.5 ng/mL in whole milk. This fluorescent latex particle immunoassay will be utilized for the detection of SEB in various foods matrices.  相似文献   

3.
The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples ( approximately 3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).  相似文献   

4.
Two competitive immunoassays, a laboratory assay based on microwell plates and a field test based on the use of polystyrene tubes, have been developed for the quantification of thiram in lettuces. Concerning the laboratory assay, the calibration curve for thiram had a linear range of 11 to 90 ng/mL and a detection limit of 5 ng/mL. Precision of the assay presented coefficient of variation values <9% and the recovery of thiram from lettuce averaged 89% across the range of the immunoassay method using 30 min extraction with water/acetone (50:50, v/v). The tube-based method was developed in order that an extract of lettuce, containing thiram at the MRL (8 ppm), would be found on the linear part of the standard curve. The calibration curve for thiram has a linear range of 100 to 800 ng/mL (1.39 to 11.1 ppm in lettuce) and a detection limit of 40 ng/mL.  相似文献   

5.
High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).  相似文献   

6.
The development of a sensitive flow-though immunosensor for the analysis of the mycotoxin zearalenone in cereal samples is described. The sensor was completely automated and was based on a direct competitive immunosorbent assay and fluorescence detection. The mycotoxin competes with a horseradish-peroxidase-labeled derivative for the binding sites of a rabbit polyclonal antibody. Control pore glass covalently bound to Prot A was used for the oriented immobilization of the antibody-antigen immunocomplexes. The immunosensor shows an IC(50) value of 0.087 ng mL(-1) (RSD = 2.8%, n = 6) and a dynamic range from 0.019 to 0.422 ng mL(-1). The limit of detection (90% of blank signal) of 0.007 ng mL(-1) (RSD = 3.9%, n = 3) is lower than previously published methods. Corn, wheat, and swine feed samples have been analyzed with the device after extraction of the analyte using accelerated solvent extraction (ASE). The immunosensor has been validated using a corn certificate reference material and HPLC with fluorescence detection.  相似文献   

7.
An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d(3)-imidazole (4-MI-d(3)) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r(2)) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6-105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8-99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.  相似文献   

8.
The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.  相似文献   

9.
Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 microg/kg) and lower than the detection limits of the usual chromatographic detection methods.  相似文献   

10.
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.  相似文献   

11.
This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.  相似文献   

12.
A monoclonal antibody-based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay were compared with those from high-performance liquid chromatography (HPLC). Three sets of sample extracts containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with enzyme-linked immunosorbent assay (ELISA) for beef liver samples gave an r(2) = 0.98 despite rather low ractopamine concentrations (range 1.1-13.4 ng/mL, n = 6). Ractopamine concentrations in cow urine samples treated by solid phase extraction, to remove ractopamine metabolites, also showed a high correlation between the HPLC and the ELISA results (r(2) = 0.95, range 1.0-275 ng/mL, n = 61). In contrast, HPLC and ELISA analyses of ractopamine in sheep urine were not well-correlated (r(2) = 0.58, range 0.85-51 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes, ELISA and HPLC methods were highly correlated [r(2) = 0.94 for sheep (range 123-10 554 ppb, n = 60) and an r(2) = 0.98 for cattle (range 14-8159 ppb, n = 62)]. Tissues contained only minute amounts of ractopamine, and after 7-day withdrawal periods, less than 1 ppb of free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples before and after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody-based ELISA could be useful for a sensitive, quantitative, or qualitative ractopamine screening assay.  相似文献   

13.
A competitive indirect enzyme-linked immunosorbent assay was developed for the fungicide iprovalicarb, using a polyclonal antibody produced against a hapten conjugated through the carboxyl group on the benzene ring to keyhole limpet hemocyanin. Under an optimized condition using a heterologous format, an IC(50) of 3.51 ng/mL and the lowest detection limit of 0.065 ng/mL were obtained. When the isopropoxy group was removed from the iprovalicarb structure for the synthesis of a hapten, the resulting hapten was not successful as an immunogen, indicating that the isopropyl moiety was an important epitope, as evidenced by the cross-reactivities of some structurally related compounds. When applied to the real crop and water samples, the recoveries were in the range of 80.52-144.70% (n = 4) and 72.11-100.43% (n = 4), respectively. Accordingly, this ELISA can be used as a useful method for monitoring iprovalicarb residues in crop and water samples.  相似文献   

14.
Mycophenolic acid (MPA) was reacted with N-hydroxysuccinimide and conjugated to keyhole limpet hemocyanin (KLH), and to horseradish peroxidase (HRP), respectively. The MPA-KLH was used to produce anti-MPA antiserum in rabbits. A competitive direct enzyme immunoassay (EIA) for MPA was established with anti-MPA antiserum and MPA-HRP conjugate. The mean 50% inhibition and detection limit of MPA standard curves (n = 103) were 197 +/- 67 and 81 +/- 48 pg/mL, respectively. The EIA was specific for MPA and its synthetic 2-morpholinoethyl ester, mycophenolate mofetil (91% relative cross-reactivity). Raw bulk milk and pasteurized milk, with and without beta-glucuronidase pretreatment, were analyzed by EIA. No MPA was found in milk, at a detection limit of 100 pg/mL (recovery 58-66% at 0.125-2 ng/mL). Blue-veined cheese from the German market (n = 53) was analyzed by EIA, and the detection limit was at 0.5 ng/g (recovery 68-79% at 5-100 ng/g). All but two cheeses contained MPA, although mostly (66%) at levels of <10 ng/g. MPA at 400-1200 ng/g was found in Roquefort cheeses. Highest levels (4-11 microg/g) were found in a German soft cheese preparation. MPA levels in mycelium-rich parts of cheese were 3 times higher than in mycelium-free parts.  相似文献   

15.
To develop a competitive indirect enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of the sulfonylurea herbicide bensulfuron-methyl, seven structurally related haptens were synthesized. Four of them mimicking the target analyte were conjugated to keyhole limpet hemocyanin by the N-hydroxysuccinimide activated ester method to use as immunogens, and all of them were conjugated to bovine serum albumin to use as plate-coating antigens. Polyclonal antibodies raised in rabbits and the coating antigens were screened and selected for the assay in simple homologous and heterologous ELISA formats. Three sensitive heterologous ELISAs were selected and optimized, showing the average IC(50) values of bensulfuron-methyl as low as 0.17, 0.09, and 0.09 ng/mL, the detection ranges of 0.04-0.60, 0.01-0.60, and 0.04-0.25 ng/mL, and the lowest detection limits of 0.03, 0.002, and 0.03 ng/mL, respectively. The cross-reactivities of other sulfonylurea herbicides and metabolites of bensulfuron-methyl to the antibodies were less than 15% in the two assays. Recoveries from the analyte-fortified water samples in assay I were in the range of 81-125% by simple dilution. The correlation between the ELISA and HPLC was 0.999 (n = 15) with a slope of 1.37 in the analysis of groundwater samples fortified with bensulfuron-methyl. The results obtained strongly indicate that the ELISA can be a highly sensitive and convenient tool for detecting bensulfuron-methyl residues in agricultural and environmental samples.  相似文献   

16.
The applicability of an ELISA for detection and quantification of benalaxyl in red wine samples is described. The study of the influence of this matrix on the reliability of the assay indicates that red wine samples require a rapid and simple cleanup step before ELISA assay. Recovery and precision of the method were evaluated by spiking red wine samples with benalaxyl in the 0.5-24 ng/mL range. Benalaxyl can be determined with good accuracy and precision up to 0. 5 ng/mL in starting red wine samples (detection limit of 0.13 ng/mL). No false negative or positive results were obtained. Authentic red wine samples were analyzed by ELISA and by RP-HPLC. The amounts of benalaxyl found by ELISA were in good agreement with RP-HPLC analysis.  相似文献   

17.
A sensitive enzyme immunoassay for cephalexin (CEX) was developed using the rabbit antiserum to CEX, beta-D-galactosidase-labeled CEX, and a double-antibody separation method. The immunogen of CEX was prepared by coupling the amino group of CEX to thiol groups introduced into bovine serum albumin by the use of N-(m-maleimidobenzoyloxy)succinimide as a cross-linker. Highly titered antiserum to CEX was produced in rabbits immunized with the immunogen. Enzyme labeling of CEX with beta-D-galactosidase was done by using N-(gamma-maleimidobutyryloxy)succinimide as the cross-linker. The limit of detection was 30 ng CEX/mL sample solution. Application of the method to CEX drug residues detected 30 ng/mL in milk, 60 ng/g in egg yolk, and 400 ng/g in hen tissue.  相似文献   

18.
A method has been developed for determining ivermectin in 5 mL samples of cattle blood by a 2-step process: cleanup solvent extraction followed by direct injection onto a normal phase liquid chromatography (LC) system with UV detection. Recovery was 77-80% +/- 5.5% standard deviation. Endogenous interference that may be present caused the lower limit of detection to be set at 4-5 ng/mL. The method was used to show that in blood the distribution of ivermectin favors plasma in a fixed proportion over cellular material, and further to provide a time-course profile of ivermectin in the whole blood of injected cattle. In whole blood, ivermectin concentration peaked between 3 and 5 days and dissipated slowly with a half-life of 3 days.  相似文献   

19.
A competitive enzyme-linked immunosorbent assay (ELISA) for pentachloronitrobenzene (PCNB), a fungicide and chemical intermediate, was developed using a polyclonal antiserum produced against a hapten-protein conjugate of pentachlorophenoxypropionic acid-bovine serum albumin (BSA). An indirect competitive ELISA of PCNB showed an IC50 of 37 ng/mL and a limit of detection (LOD) of 7 ng/mL. The ELISA can tolerate up to 10% (v/v) methanol, 5% (v/v) acetonitrile, or 5% (v/v) acetone without significant fluctuation of Amax and IC50. The assay sensitivity showed little change in a range of pH from 6 to 8 and concentrations of 0.05-0.2 M NaCl in the assay buffer. Very low cross-reactivities were observed for some structurally related compounds except for hexachlorobenzene (12%). The average recoveries of PCNB from fortified well water, river water, and soil samples were in ranges of 88-94, 80-91, and 70-81%, respectively. The correlations between the gas chromatographic and ELISA results were excellent (r 2 >or= 0.97, slopes from 0.86 to 1.10) for those fortified samples. The ELISA is a good alternative tool for monitoring PCNB residues in environmental samples.  相似文献   

20.
Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.  相似文献   

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