Development of an enzyme-linked immunosorbent assay for the detection of the fungicide fenarimol

To develop an enzyme-linked immunosorbent assay for the fungicide fenarimol, two synthesized haptens, haptens-1 and -2, and the purchased 4,4'-DDA were conjugated to carrier proteins (BSA, KLH, and OVA). Polyclonal antibodies raised against hapten-1,2-KLH conjugates in rabbits and the coating antigens of hapten-1,2-BSA conjugates, hapten-2-OVA conjugate, and 4,4'-DDA-BSA conjugate were screened and selected for the homologous and/or heterologous ELISA formats. Two competitive indirect ELISAs were selected: assays I and II. The optimized ciELISAs of assays I and II showed average IC(50) values of fenarimol of 5.4 and 9.4 ng/mL, detection ranges of 1.1-25.9 and 1.1-82.7 ng/mL, and lowest detection limits of 0.3 and 0.3 ng/mL, respectively. The cross-reactivities with several structurally related compounds indicated the importance of the steric fitness in the antigen-antibody interaction. Recoveries of fenarimol from apple and pear samples spiked with the analyte by assay I were in the range of 93-113% by simple extraction, concentration, and dilution. This assay could be a convenient and supplemental analytical tool for monitoring fenarimol residues in environmental and agricultural samples.     

Production of the polyclonal antibody against Sudan 3 and Immunoassay of Sudan dyes in food samples

In this study, 4-aminophenylacetic acid was covalently coupled to aniline to synthesize an intermediate hapten and the intermediate hapten was coupled to β-naphthol to synthesize a tentative hapten of Sudan 3. The hapten was coupled to bovine serum albumin as the immunogen to produce the polyclonal antibody. The obtained antibody was highly specific to Sudan 3, Sudan 1, and Para red, but showed relative low binding ability to Sudan 2, Sudan 4, and Sudan red G. After evaluation of different coating antigens, a heterologous indirect competitive immunoassay was developed to multidetermine the six red dyes in food samples. The cross reactivities to the six analytes were in a range of 21-105%, and the limits of detection were in a range of 0.1-0.8 ng/mL depending on the compound. Intra- and interassay recoveries from the standard fortified blank samples were in a range of 74.5-96.3% with coefficients of variation lower than 15.1%.     

Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the organophosphorus insecticide acephate, O,S-dimethyl acetylphosphoramidothioate, was developed using a polyclonal antibody. Five different haptens mimicking the analyte were synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) by the N-hydroxysuccinimide active ester and diazotization methods. Polyclonal antibodies raised against hapten-KLH conjugates in rabbits and hapten-BSA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems. The effects of various assay conditions such as detergent, organic solvents, pH, and preincubation of the mixture of the polyclonal antibody and the analyte on the sensitivity were evaluated. The IC(50) value for acephate was 25 ng/mL in an optimized heterologous system using hapten-4-BSA as a coating antigen and a polyclonal antibody no. 8377 against hapten-1-KLH, showing the detection range of 5-140 ng/mL and the lowest detection limit of 2 ng/mL. The cross-reactivities of the structurally related organophosphorus insecticides, including the major metabolite of the analyte, methamidophos, were less than 1%. Recoveries from the analyte-fortified tap water, mulberry leaves, and lettuce samples in the assay were in the range of 72-121% by simple extraction, concentration, and dilution. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring acephate residues in environmental and agricultural samples.     

Hapten syntheses and antibody generation for a new herbicide, metamifop

To develop a competitive indirect enzyme-linked immunosorbent assay for metamifop, a new aryloxyphenoxypropionic acid herbicide, three structurally related haptens were synthesized. Hapten conjugates to keyhole limpet hemocyanin and bovine serum albumin were used as immunogens and plate-coating antigens, respectively. Various sets of polyclonal antibodies from rabbits and the coating antigens were screened for the assay in simple homologous and heterologous ELISA formats. A selected heterologous ELISA was optimized to show an average IC50 value as low as 20.1 ng/mL, detection ranges of 1.0-350 ng/mL, and a lowest detection limit of 0.1 ng/mL. The cross-reactivities of other aryloxyphenoxypropionic acid herbicides to the antibodies were less than 0.5% in the assays except fenoxaprop-P and fenoxaprop-P ethyl, having a diaryl ether group identical to that of metamifop. Molecular modeling studies revealed that the physicochemical properties of the diaryl ether group are the most important determinants of sensitivity and selectivity. The results strongly indicate that the selected set of ELISA is a highly sensitive and convenient tool for detecting metamifop.     

Enzyme-linked immunosorbent assay for the pyrethroid permethrin

Permethrin is a predominant pyrethroid widely used in agriculture and public health. A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of permethrin was developed. Two haptens, the trans- and cis-isomers of 3-(4-aminophenoxy)benzyl-3-(2, 2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The resulting ELISA has an I(50) value of 2.50 microg/L and relatively low cross-reactivities with other major pyrethroids, such as esfenvalerate, cypermethrin, deltamethrin, and cyfluthrin. Methanol was found to be the best solvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters are unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths (>0.2 M PBS) strongly suppress the absorbances. River water samples fortified with permethrin were analyzed according to this method and validated by GC-MS. Good recoveries and correlation with spike levels were observed, suggesting this immunoassay is valuable for environmental monitoring and toxicological studies at parts per trillion levels of permethrin.     

Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect ciprofloxacin (CPFX) in food animal edible tissues. CPFX was converted by an active ester method into conjugates CPFX-bovine serum albumin (CPFX-BSA) and CPFX-human serum albumin (CPFX-HSA), which both allowed production of CPFX-specific rabbit antisera. In the ELISA, CPFX-HSA was coated onto the microtiter plate, followed by incubation with standard CPFX and anti-CPFX antibody. The indirect competitive ELISA revealed that the antisera have no cross-reactivity with penicillin, gentamicin, neomycin, sulfadiazine, and chlortetracycline. The antisera cross-reacted with enrofloxacin and norfloxacin about 69.8 and 44.6% as much as they did with CPFX. This ELISA was highly sensitive (0.32 ng/mL) to CPFX determination. Recovery of CPFX at 40 microg/kg was 75.58% in pork, 81.29% in chicken, and 84.30% in milk. The coefficients of variation varied from 3.7 to 9.2% over the range of CPFX concentrations studied. The linear detection range was between 1.6 and 1000 ng/mL. The results suggest that this ELISA is a specific, accurate, and convenient method for the detection of CPFX residues in food animal edible tissues.     

Development of an immunoassay for the residues of the herbicide bensulfuron-methyl

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.     

Production and characterization of monoclonal antibodies against the antibiotic tilmicosin

Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocalization of tilmicosin.     

Enzyme-linked immunosorbent assay for the pyrethroid deltamethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate and 3-[(+/-)-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropan ecarbonyloxy]methyl]phenoxyacetic acid, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized. The I(50) for deltamethrin was 17.5 +/- 3.6 microg/L, and the lower detection limit was 1.1 +/- 0.5 microg/L. This ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin, phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

Hapten synthesis and development of polyclonal antibody-based multi-sulfonamide immunoassays

This paper reports the synthesis of five sulfonamide derivatives, the production of broad-specificity polyclonal antibodies for immunoassay of sulfonamides, and the analysis of milk samples by developed assay. The three-step synthesis procedure reported in most of the literature was adopted and modified in this study. In the procedure, the purification of the intermediate was avoided and the time of synthesis was shortened from >20 to 6-9 h with improved yields. This method is generally applicable to the synthesis of haptens containing the common structure of sulfonamides. Three haptens were coupled to keyhole limpet hemocyanin, and polyclonal antibodies were obtained from rabbits immunized with these conjugates. Using the antibodies obtained, from one of these was developed an enzyme-linked immunosorbent assay (ELISA) based on the competition between free sulfonamides and the hapten-horseradish peroxidase (HRP) conjugates. The hapten-HRP conjugate giving the best competitive results and 11 structurally different sulfonamides showed 50% inhibition at concentrations of <100 ng mL(-1). After removal of the protein with acetone, milk samples were analyzed by ELISA directly; a matrix effect could be avoided when a 1:20 dilution with phosphate-buffered saline was used, and 104-131% recoveries of spiked samples were obtained. The developed immunoassay is suitable to determine sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine, sulfapyridine, and sulfamethizole below the maximum residue limit in milk (100 ng mL(-1) of total sulfonamides) rapidly and reliably.     

Synthesis of haptens for development of antibodies to alkylphenols and evaluation and optimization of a selected antibody for ELISA development

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for a class of endocrine disrupting compounds, 4-nonylphenol, is described. The parent molecule was derivatized at the ortho position of the free phenolic hydroxyl group to obtain the hapten, NP1, and it was conjugated with keyhole limpet hemocyanin, which was used as an immunogen. Four antisera were generated and screened against three coating antigens. The most sensitive ELISA from the screening tests (antiserum NP03As, 1/1000, and coating antigen NP1-BSA, 1 microg/mL) was further optimized and characterized. The influence of various physicochemical factors (organic solvent, pH, ion strength) was investigated. Methanol as the additive organic solvent was found to be the best organic solvent for the ELISA, with optimal sensitivity observed at a concentration of 5%. The ELISA parameters were changed at more acidic or basic pH values, whereas higher ionic strengths strongly suppressed the I(50) value and the maximum absorbance. The most sensitive ELISA for 4-nonylphenol exhibited an I(50) value of 38.6 +/- 5.5 microg/L, with a dynamic range from 12 to 350 microg/L, and the lower limit of detection was 7.7 +/- 1.3 microg/L. The optimized ELISA displayed no significant cross-reaction against the parent compounds, nonylphenol ethoxylates, degradation products, carboxylates, and bisphenol A, except in 4-octylphenol.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid cypermethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of cypermethrin was developed. Two haptens, the trans- and cis-isomers of 3-[(+/-)-cyano-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyloxy]methyl]phenoxyacetic acid, were conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for cypermethrin was optimized and characterized. The IC(50) for cypermethrin was 13.5 +/- 4.3 microg/L, and the lower detection limit (LDL) was 1.3 +/- 0.5 microg/L. This ELISA had relatively low cross-reactivities with other major pyrethroids, such as deltamethrin, phenothrin, resmethrin, fluvalinate, and permethrin. Methanol was found to be the best organic cosolvent for this ELISA, with an optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was applied to various domestic and environmental water samples. The water samples, fortified with cypermethrin, were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.     

Production and specificity of polyclonal antibodies to hexanal-lysine adducts

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.     

Development of an enzyme-linked immunosorbent assay for the detection of pentachloronitrobenzene residues in environmental samples

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.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid insecticide cyhalothrin

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of the pyrethroid insecticide cyhalothrin. Three haptens with an amine or propanoic acid terminus were synthesized and then conjugated with bovine serum albumin to give immunogens. Eight polyclonal antisera produced by rabbits were screened for titers and affinity using three different coating antigens. The antiserum CWB-C had the highest affinity with cyhalothrin and a low affinity with fenvalerate, fenpropathrin, deltamethrin, and fluvalinate. The half-maximum inhibition concentration for cyhalothrin was 37.2 microg/L, and the limit of detection was 4.7 microg/L. The recoveries of different concentrations of cyhalothrin (0.1-2500 microg/L) from fortified tap water, well water, and wastewater samples as determined with the ELISA were 81-114%.     

Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals

To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.     

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.     

Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol

Zilpaterol is an beta-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLH resulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigen for ELISA development. The average IC(50) derived from the developed zilpaterol immunoassay was 3.94 +/- 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists. The assay was tolerant of up to 10% (v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrations ranging from 0.05 to 1.0 M minimally affected B(0) or IC(50) values. When buffer pH was <7 or >8.8, the IC(50) values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specific zilpaterol ELISA has been developed that can serve as a rapid screening assay.     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

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.