Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

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.     

Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.     

Development of a magnetic particle-based enzyme immunoassay for the determination of penoxsulam in water

A competitive enzyme-linked immunoassay (ELISA) for the quantitation of Penoxsulam [2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide] in ground and surface waters was developed. This immunoassay utilizes magnetic particles as the solid phase to which polyclonal rabbit anti-Penoxsulam antibodies are attached. The ELISA has an estimated detection limit of 0.17 ppb (microg/mL) of Penoxsulam in water. Specificity studies indicate that the antibody can distinguish Penoxsulam from its major metabolites and structurally similar pesticides. Interference studies indicate that the ELISA has a wide tolerance of sample pH and salinity and for compounds commonly found in surface and ground waters. The ELISA was shown to compare favorably to LC-MS/MS on ground and surface water samples (r(2) = 0.957). The various studies performed demonstrate the usefulness of the ELISA technique as a rapid and high-throughput analytical method for the cost-effective monitoring of water samples.     

A miniaturized curcumin method for the determination of boron in solutions and biological samples

Methods for the determination of boron require either sophisticated and expensive equipment (e.g. ICP-OES or ICP-MS) or a relatively large sample volume (e.g. spectrophotometric methods). A modified and miniaturized spectrophotometric curcumin method is presented which allows the determination of boron in sample volumes of 50—150 μl with a detection limit of 0.010 mg B l¹. The calibration curve is linear up to a concentration of 3 mg B l¹. Interferences with other ions are eliminated by extraction of boron with 2-ethyl-1,3-hexanediol in chloroform. Simultaneously, enrichment of boron in the sample can be performed within the same extraction step. Results show good reproducibility with a relative standard deviation of 15% at B concentrations between 0.05 and 0.40 mg B l^—1. In different types of solutions, recovery of added boron was in the range of 98 to 99%. For several plant materials containing from 18 to 58 μg B (g dw)¹, an overall relative standard deviation of 4.5% was established. So far, the method has been successfully applied to determine boron in waters, nutrient solutions, different plant parts (roots, stems and leaves) as well as in phloem sap and apoplastic washing fluid of several plant species.     

Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).     

Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides

A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides.     

Simultaneous gas chromatographic determination of dibutyltin and tributyltin compounds in biological and sediment samples

A method is described for the simultaneous determination of nanogram amounts of dibutyltin and tributyltin compounds in biological and sediment samples. These compounds are converted to the corresponding chlorides with HCl, extracted with ethyl acetate-hexane (3 + 2) for biological samples and with hexane for sediment samples, and hydrogenated with sodium borohydride. The corresponding hydrides, Bu2SnH2 and Bu3SnH, are detected by electron-capture gas chromatography after cleanup by silica gel column chromatography. Detection limits are 1.0-2.0 and 0.5-1.0 ng/g, respectively, for biological and sediment samples.     

Preparation of pentachlorophenol derivatives and development of a microparticle-based on-site immunoassay for the detection of PCP in soil samples

Pentachlorophenol (PCP) is used as a herbicide in agriculture and as an insecticide for termite control. Because of the apparent hazard associated with its usage, there is a need for an efficient and economic on-site screening method. A 5-min on-site test has been developed for the detection of PCP based on the OnTrak format, a successful Roche on-site test format for drugs of abuse, utilizing the principle of latex agglutination immunoassay. The test detects 1 ppm of PCP in soil samples.     

Rapid determination of fumonisin B1 in food samples by enzyme-linked immunosorbent assay and colloidal gold immunoassay

A rapid enzyme-linked immunosorbent assay (ELISA) test (microwell plate) and a membrane-based colloidal gold immunoassay in flow-through and lateral-flow formats for the rapid detection of fumonisin B1 (FB1) were developed. The rapid microwell assay can be completed within 20 min with the detection limit of 0.5 +/- 0.2 microg/L. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 microg/L for FB1 with the detection time of <10 min. Matrix interference was eliminated by 15-fold dilutions of methanol extracts with buffer. These immunoassays can be used as quantitative or qualitative tools for the rapid detection of FB1 residues in 10-20 min on-site.     

High-volume enzyme immunoassay test system for sulfamethazine in swine

A high-volume enzyme immunoassay (EIA) system for slaughterhouse screening of sulfamethazine in swine plasma/serum has been developed. The system includes a robotic sample processor that performs most of the liquid handling requirements in the assay. The assay gives good correlation with the widely used thin layer chromatographic method for determination of sulfamethazine in serum and plasma (r = 0.826). Inter- and intra-assay coefficients of variation are less than 10%. Approximately 2,400 serum/plasma samples can be analyzed in a normal working day (8 h) with this system.     

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm(-)(1)) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.     

Enzyme immunoassay for determination of gluten in foods: collaborative study

A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods.     

Aflatoxin, a human carcinogen: determination in foods and biological samples by monoclonal antibody affinity chromatography

We have used monoclonal antibody technology to produce antibodies that recognize aflatoxins in order to develop noninvasive methods in conjunction with other chemical analytical techniques to monitor human exposure to environmental carcinogens. These methods require the ability to quantitate aflatoxins and their metabolites, including DNA and protein adducts, in readily accessible compartments such as serum and urine. The techniques permit efficient analysis of many samples in a relatively short time. Also, these monoclonal antibody affinity columns have been extremely useful for rapid isolation of aflatoxins from food and grain samples, as well as aflatoxin M1 from milk. Monoclonal antibody affinity methods are nondestructive to the aflatoxin molecule, so the sample aliquot can be used for confirmation. The use of monoclonal antibody preparative affinity columns represents a major, substantive breakthrough for analytical chemists and will be a generally applicable technology for isolation of many different substances.     

Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study

A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.     

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.     

Development and validation of an ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M1 in milk

A fast and ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M(1) in milk samples has been developed and validated. The method is an indirect competitive type format involving the immobilization of an aflatoxin M(1)-bovine serum albumin conjugate on 384 well black polystyrene microtiter plates and the use of a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate. Aflatoxin M(1) standard solutions were prepared in milk-based buffer, and milk samples were analyzed without any cleanup procedure. The limit of quantification was 1 ppt, the coefficient of variation was below 9% for both intra- and interassay precision, and the recovery ranged from 96 to 122%. The method is specific, and other aflatoxins do not significantly cross-react with the antibody. Twenty-four milk samples were analyzed, and a good correlation was observed (y = 0.98x + 1.71, r(2) = 0.98, n = 24) when the data were compared with a reference high-performance liquid chromatography method with a fluorescent detector. The developed method is suitable for an accurate, sensitive, and high-throughput screening of aflatoxin M(1) in milk samples with a reduction of costs and increased detectability, as compared with previously developed immunoassays.     

A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.     

Digestion of food samples for total mercury determination

A method of digestion by using a mixture of hydrochloric, nitric, and sulfuric acids has been developed for the determination of total mercury in a wide range of food samples. Good recoveries of mercury were obtained from NBS (National Bureau of Standards) Albacore Tuna and from food samples spiked with inorganic mercury. A detection limit of 0.01 microgram mercury/g can be obtained.