Hapten synthesis and monoclonal antibody-based immunoassay development for detection of the fungicide trifloxystrobin

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).     

Determination of ochratoxin A by monoclonal antibody-based enzyme immunoassay

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl

Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase.     

Characterization of monoclonal antibodies for lead-chelate complexes: applications in antibody-based assays

Monoclonal antibodies against lead were generated by immunizing BALB/c mice with lead conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, S-2-(4-aminobenzyl)diethylenetriamine pentaacetic acid (DTPA). Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. One of the hybridomas generated from this fusion (4/7) synthesized and secreted an antibody that bound tightly to Pb2+-DTPA complexes but not to metal-free DTPA. The performance for a competitive inhibition enzyme-linked immunosorbent assay (ELISA) incorporating this antibody was assessed for its sensitivity to changes in pH, ionic strength, and blocking reagents. The cross-reactivities in this ELISA were less than 3% for Fe3+, Cd2+, Hg2+, and Cu2+ and less than 0.3% for Cr3+, Mn2+, Mg2+, In3+, Ag1+, Ni2+, Co2+, Zn2+, Ca2+, Cu1+, and Hg1+. The IC50 value achieved for lead was 2.72 +/- 0.034 microM, showing the detection range of 0.092-87.2 microM and the lowest detection limit of 0.056 +/- 0.005 microM. Recoveries from the analyte-fortified tap water and ultrapure water were in the range of 80-114% . These results indicate that the ELISA could be a convenient analytical tool for monitoring lead residues in drinking water.     

Development of monoclonal ELISAs for azinphos-methyl. 1. Hapten synthesis and antibody production.

The development of monoclonal antibody-based enzyme-linked immunosorbent assays for azinphos-methyl is described. A panel of haptens was synthesized for immunoconjugate preparation, and a series of haptens for heterologous, coating or tracer, conjugates was also prepared. Hapten synthesis was based on a strategy in which only a fragment of the whole target molecule was present (fragmentary haptens). From immunized mice, a set of monoclonal antibodies was obtained and ELISA sensitivities were assayed in different formats. Affinities estimated as I(50) values in the low nanomolar range for azinphos-methyl and phosmet were observed for several monoclonal antibodies in the conjugate-coated format and in the antibody-coated format under nonoptimized assay conditions.     

Hapten synthesis and monoclonal antibody-based immunoassay development for the detection of the fungicide kresoxim-methyl

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.     

Development of a monoclonal antibody-based enzyme-linked immuosorbent assay for the beta-adrenergic agonist zilpaterol

Zilpaterol is a beta-adrenergic agonist approved for use as a growth promoter in cattle in South Africa and Mexico but not in the European Union, United States, or Asia. Here, we report the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for zilpaterol. Mice immunized with zilpaterol-butyrate-keyhole limpet hemocyanin were utilized for monoclonal antibody generation whereas zilpaterol-butyrate-bovine serum albumin was used as a coating antigen for ELISA. Thirteen clones were isolated, and after the initial sensitivity and isotyping experiments, three clones were selected for further ELISA optimization. Studies indicated that the optimum pH was near 7.4. Clone 3H5 had the highest sensitivity to zilpaterol and some interaction with clenbuterol and terbutaline at high concentrations but not other N-alkyl [bamethane, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists tested. However, clone 3H5 was not functional at high salt concentrations, which precluded further development for urine analysis. Clone 2E10 showed increased sensitivity as salt concentrations were increased and did not cross-react with any of the structural analogues tested. However, its sensitivity to salt and urine concentration changes could cause high variability. Clone 7A8 showed good sensitivity and only a modest change with the salt concentration changes. Clone 7A8 also demonstrated smaller changes in IC(50) and B(0) with increasing sheep urine or cattle urine concentrations as compared to clones 2E10 or 3H5 and, thus, was selected for further development. The IC(50) for all of the antibodies showed exponential increases with increasing organic solvents concentrations, making it desirable to minimize solvent levels. In conclusion, a sensitive, specific zilpaterol monoclonal antibody-based ELISA has been developed that can serve as a rapid screening assay.     

Development of monoclonal antibody-based enzyme-linked immunosorbent assay for gossypol analysis in cottonseed meals

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the analysis of gossypol in cottonseed meals. First, the checkerboard method was used to determine the optimum amount of coating antigen gossypol-BSA (bovine serum albumin) and primary anti-gossypol monoclonal antibody (Mab) needed in the ic-ELISA. Second, the effects of several physical (incubation time and temperature) and chemical (solvent types and concentrations) conditions on the performance of Mab on ic-ELISA were investigated to get a rapid robust assay with high sensitivity. Under the established optimized condition, the concentration of gossypol giving 50% reduction of the maximum ELISA signal (I50) in the competitive standard curve was 0.20 microg/mL, whereas the detection limit for gossypol was 0.024 microg/mL. This ic-ELISA method for the analysis of gossypol extracted by methanol from a variety of cottonseed meals was further compared with the official method of the American Oil Chemists' Society (AOCS). The amounts of gossypol determined by the ic-ELISA had a good correlation with those obtained by the AOCS method (R2 = 0.90).     

Rapid monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Listeria in food products

A rapid diagnostic test for the detection of Listeria in food products has been created. This test, known as Listeria-Tek, uses 2 monoclonal antibodies specific for Listeria in an enzyme-linked immunosorbent assay (ELISA) format. The test requires only 40 h of broth enrichment with no culturing on solid media. It is extremely simple to perform and easy to interpret, and is at least as sensitive and accurate as the best of the culture methods. The test can be used with dairy products, meat products, and environmental samples. The ELISA test is safely performed on the open bench of the laboratory because no live cultures, no radioactivity, no phage, etc., are necessary. There is no need for special licenses or reserved laboratory space, and no waste disposal problems are encountered. If necessary, one technician could easily perform hundreds of assays per day. A printed data sheet is available for permanent records.     

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.     

Development of monoclonal antibody-based immunoassays to the N-methylcarbamate pesticide carbofuran

To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently characterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterologous assay haptens. Depending on the immunoreagent combination and assay format, competitive assays with I(50) values in the 1.2-10.2 nM (0.27-2.27 ng/mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFNH, coupled either to horseradish peroxidase or to ovalbumin, was used to develop a direct and an indirect enzyme-linked immunosorbent assay, respectively. Optimized immunoassays displayed very similar analytical characteristics, with an I(50) value around 0.7 ng/mL and a limit of detection around 0.08 ng/mL. Both immunoassays were able to tolerate the presence of methanol up to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pesticides were not recognized by the MAb. These immunoassays should reasonably allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practical importance.     

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.     

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals

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.     

Monoclonal antibody-based fluorescence polarization immunoassay for sulfamethoxypyridazine and sulfachloropyridazine

In this paper, a new monoclonal antibody (Mab) against sulfamethoxypyridazine (SMP) was produced, and a fluorescence polarization immunoassay (FPIA) based on the produced Mab was developed and optimized for the qualitative screening analysis of SMP. The Mab was raised from mice immunized with SMP linked to bovine serum albumin (BSA) by carbodiimide activated ester formation, using a succinic anhydride spacer molecule between SMP and BSA. Fluorescein labeled sulfachloropyridazine (SCP) and SMP (tracer) were synthesized and purified by thin layer chromatography (TLC). The developed screening FPIA method can tolerate up to 20% methanol, and satisfactory assay sensitivity can be obtained between pH 4 and pH 8 and at lower salt concentration. The anti-SMP Mab exhibited a high cross-reactivity with SCP. The effect of the tracer structure on the analytical characteristic of the determination and on antigen-antibody binding constants was studied. The limits of detection (LOD) were 0.7 ng/mL for SMP and 0.25 ng/mL for SCP in buffer, respectively, whereas negligible cross-reactivities were exhibited by related sulfonamides. Analysis of SMP and SCP-fortified milk samples by the FPIA showed average recoveries from 60 to 145%.     

Uptake and xylem transport of fipronil in sunflower

The phenylpyrazole insecticide, fipronil, is used in seed coating against Agriotes larvae, which infest mainly corn and sunflower. Coating the seeds of the cultivated plants with fipronil has proven its effectiveness against Agriotes populations. In the case of sunflower or even corn, the possible root uptake of this insecticide may lead to a toxic effect against pollinators such as honeybees. In the present report, the uptake and transport of fipronil inside the sunflower seedling was studied in the laboratory. In a first study, sunflower was cultivated on an aqueous medium containing fipronil. An intense root uptake of fipronil occurred, leading to a transport into leaves depending upon transpiration. In a second study, plants were cultivated on a soil in which fipronil was uniformly distributed. Under our soil conditions (20% organic carbon), the partition coefficient between soil and water (K(d)) was found to be equal to 386 +/- 30. The average rate of fipronil transfer from soil water to seedlings was from 2 to 2.6 times lower than water transfer. During the 3 week experiment, 55% of recovered labeled compounds was in the parent form and 35% had been converted to lipophilic metabolites, with either a 4-CF(3)-SO(2) or 4-CF(3)-S substituant, which are also very potent lipophilic insecticides. This paper suggests that the possible uptake of fipronil by sunflower seedlings under agronomic conditions is mainly controlled by the physicochemical characteristics of the seed-coating mixture.     

Transformation and sorption of fipronil in urban stream sediments

Fipronil is an urban-use insecticide, and the increased use has led to its frequent detections in urban streams. Most studies on the environmental fate of fipronil so far have focused on soils, and little is known about its behavior in sediment-water systems. In this study, we investigated the transformation and sorption of fipronil in urban stream sediments from California, incubated under facultative and anaerobic conditions. Degradation of fipronil in sediments generally followed exponential decay kinetics, and the first-order half-lives of fipronil were only 4.6-18.5 days in anaerobic sediments. The persistence of fipronil under facultative conditions was considerably longer, with half-lives from 25 to 91 days. Sterilization generally decreased the dissipation of fipronil, indicating that microbial activity was an important factor in fipronil transformations in sediments. Under facultative conditions, fipronil sulfide and sulfone were observed, while only fipronil sulfide was detected in anaerobic samples. The sorption coefficient K d consistently increased with organic carbon contents of sediments. In the same sediment, K d usually also increased with contact time, suggesting decreased availability for aged residues. Results from this study showed that the stability of fipronil in sediments depends closely on the oxygen status and that due to the readily conversion of fipronil to the sulfone and sulfide metabolites, the overall risk assessment of fipronil in surface aquatic systems should take into consideration fipronil as well as its metabolites.     

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 2. Evaluation of rapid extraction methods and implications for the analysis of incurred residues in chicken liver tissue

Several rapid extraction methods were evaluated for use with a monoclonal antibody-based competitive inhibition ELISA (cELISA) to detect sulfadimethoxine (SDM) in chicken liver tissue. These methods included extraction of the samples with (1) aqueous buffer with or without ultrafiltration, (2) acetonitrile/water, (3) methanol/water, or (4) acetone. The organic extraction methods were evaluated with or without solvent evaporation prior to dilution into assay buffer for the cELISA. The aqueous-based extraction methods were compatible with the cELISA. However, of the organic extraction methods, only the acetone liver extract with solvent evaporation prior to analysis was compatible with the cELISA. The cELISA method coupled to aqueous- or acetone-based sample extraction as well as an HPLC method was evaluated for the analysis of chicken liver tissues fortified with SDM at levels from 0.2 to 0.025 ppm. Mean SDM recoveries for the HPLC method and for the cELISA method using samples prepared by aqueous extraction, aqueous extraction and ultrafiltration, or acetone extraction, evaporation, and reconstitution were 68.9, 95.7, 60.1, and 52.5%, respectively. For the analysis of samples obtained from an SDM incurred residue study, HPLC and cELISA analysis of the same organic extract gave results that were highly correlated (R(2) = 0.976; p < 0.0001). However, results obtained from the analysis of aqueous extracts by cELISA did not correlate well with those obtained by HPLC (R(2) = 0.61, p > 0. 0006). This was attributed to the coextraction of cross-reactive SDM-related residues that were not quantified by the HPLC method. The presence of these residues should be considered during data interpretation when ELISA methods coupled with rapid aqueous extraction of samples are used in SDM residue monitoring programs.     

Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.     

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.