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.     

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.     

Competitive direct enzyme-linked immunosorbent screening assay for the detection of sulfamethazine contamination of animal feeds.

A sensitive screening method has been developed for detecting sulfamethazine (SMZ) contamination of feeds by using either polyclonal or monoclonal antibodies and a direct competitive enzyme-linked immunosorbent screening assay (ELISA). Feed samples of 25.0 g are extracted with 0.5N HCl and centrifuged. The extract is adjusted to pH 7.0 with 3.0N NaOH and recentrifuged. This pH-adjusted extract is used in the ELISA. Levels as low as 0.004 micrograms SMZ/g feed were detected in supplemented extracts by polyclonal antibodies; levels of 0.4 micrograms SMZ/g feed were detected by a monoclonal antibody.     

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.     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets by polyclonal antibody-based enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the species identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. The assay was performed in two different formats, microtiter plates and immunostick tubes, and uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of grouper (anti-GSP), wreck fish (anti-WSP), and Nile perch (anti-PSP). The antibodies were made species-specific by blocking them with the heterologous soluble muscle proteins. Immunorecognition of polyclonal antibodies adsorbed to their specific fish samples was made with swine antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of the species studied.     

Enhanced surveillance of foodborne mycotoxins by immunochemical assay

Mycotoxins are a chemically diverse group of fungal secondary metabolites with a wide range of toxic effects. Conventional thin-layer and instrumental methods of mycotoxin analysis are time-consuming and make routine safety and quality control screening of these compounds in agricultural commodities difficult. As an alternative, specific polyclonal and monoclonal antibodies have been raised against mycotoxin-protein conjugates and used in sensitive radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs). One of the simplest ELISA approaches involves competition for a solid-phase antibody between a mycotoxin-enzyme conjugate and an unconjugated mycotoxin in the sample extract. ELISAs have been developed for aflatoxins B1 and M1, zearalenone, T-2 toxin, and deoxynivalenol, which are highly specific, rapid (10 min), easily adaptable for analyzing large numbers of samples, and directly applicable to assaying methanol-water extracts of a wide range of foods. Several commercial mycotoxin ELISAs using this approach (most typically for aflatoxin B1) are currently being marketed. Since ELISAs will be used in large part by personnel with limited technical expertise, individual kits must be critically evaluated by analytical chemists for suggested sampling procedures, efficiency of extraction, cross-reactivity, mycotoxin recovery, assay reproducibility, and product shelf-life prior to routine use in food safety and quality control screening.     

Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin

Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.     

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.     

Production and characterization of a monoclonal anti-anti-idiotype antibody against fumonisin B(1).

A monoclonal anti-anti-idiotype antibody (mAb3) against fumonisin B(1) (FmB1) was produced from the hybridoma cell line 7C7F4, which was generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse that had been immunized with the Fab fragments of affinity-purified anti-idiotype antibodies. The mAb3 belongs to the immunoglobulin M, kappa light chain. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were established for antibody characterization and toxin analysis. In an idc-ELISA using FmB1-ovalbumin (OVA) as the coating antigen, the concentrations causing 50% inhibition of binding (IC(50)) of mAb3 to the solid-phase FmB1-OVA by free FmB1, FmB2, and FmB3 were found to be 75, 95, and 450 ng/mL, respectively. In the dc-ELISA, the concentration causing IC(50) of FmB1-horseradish peroxidase to the solid-phase mAb3 by free FmB1 was found to be 233 ng/mL. Analysis of 12 samples naturally contaminated with fumonisins with mAb3-based idc-ELISA and polyclonal-based dc-ELISA showed a good correlation between these two methods with a correlation coefficient of 0.76 at p < 0.02. The linear regression slope was found to be y[polyclonal ELISA] = 0.87x[mAb3 ELISA] - 52 ppb.     

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.     

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.     

Production of monoclonal antibodies to the N-methylcarbamate pesticide propoxur

Monoclonal antibodies (MAbs) to the pesticide propoxur were produced from haptens with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure. MAbs were subsequently characterized in the conjugate-coated format using these immunizing haptens and newly synthesized compounds as homologous and heterologous assay conjugates, respectively. Appropriate combinations of immunoreagents resulted in competitive enzyme immunoassays (ELISA) with I(50) values in the low nanomolar range (6.5-17.9 nM). A modification of the conjugate-coated format consisting of the simultaneous incubation of the MAb and the peroxidase-labeled secondary antibody in the presence of the analyte resulted in an assay with an I(50) value of 4.4 nM. This one-step conjugate-coated ELISA format is as simple and fast as the antibody-coated format but without the need of synthesizing enzyme-hapten conjugates. Major N-methylcarbamate pesticides were not recognized by the MAb. This immunoassay should reasonably allow the rapid, low-cost, and sensitive determination of propoxur in food, soils, and the environment at levels of regulatory and practical importance.     

Generation of group-specific antibodies against sulfonamides

To develop a sulfonamide-specific ELISA, different attempts were made to obtain monoclonal antibodies specific for the common structure of sulfonamides. In a first approach, sulfanilamide was linked to albumins using glutaraldehyde or a succinimide ester as cross-linker. A weak immune response or none at all was induced after immunization of mice with those conjugates. High antibody titers were obtained with conjugates where sulfanilamide was linked to albumins or casein (azocasein) with a diazotation reaction. However, the antibodies were only highly specific for the bound sulfanilamide molecule. In a second approach, sulfonamide-protein conjugates were used in which the sulfonamide molecule is linked at its side chain, leaving the common structure of sulfonamides unchanged. Three sulfonamide derivatives (S, TS, and PS, previously described in the literature) containing a carboxyl group in their side chain were linked to proteins using a carbodiimide mediated reaction. Immunization with the S-conjugates led to high antibody titers, but the antibodies were only highly specific for the bound S-molecule. Group-specific antibodies were obtained after immunization with the PS- and TS-conjugates. It was described that immunization with PS-conjugates lead to the recognition of other sulfonamides (sulfamethazine, -merazine, -diazine, and -dimethoxine) that are not well recognized by antibodies induced after immunization with TS-conjugates. Therefore, we tried to guide the immune response in the direction of recognition of the common structure of sulfonamides by immunizing the animals alternately with PS- and TS-conjugates. The polyclonal antibodies of the mice indeed had a broader specificity, but the specificity of the monoclonals obtained after fusion experiments was not influenced. Immunization with TS-conjugates seemed sufficient to obtain sulfonamide-specific monoclonal antibodies. With the best monoclonal (mAb 3B5B10E3) two competitive inhibition (ci) ELISA's were developed: one coated with antigen and the other coated with the monoclonal antibody. Sulfadiazine, -dimethoxine, -thiazole, -pyridine, and -methoxazole were detected in both ELISA's at their MRL-value (100 ppb) in buffer solution. Sulfadiazine, sulfathiazole, and sulfamethoxazole could even be detected at 10 ppb.     

Neamin as an immunogen for the development of a generic ELISA detecting gentamicin,kanamycin, and neomycin in milk

A broad-specific ELISA using one antibody preparation for the detection of gentamicin, kanamycin, and neomycin in milk is reported for the first time. For the immunization of rabbits, neamin was used as the generic hapten on the basis of the facts that it is a two-ring fragment of neomycin and, in shape and charge, it resembles parts of kanamycin and gentamicin. Neamin was linked to the preactivated carrier protein keyhole limpet hemocyanin by EDC coupling. The specificity and sensitivity of the polyclonal antibodies for the aminoglycoside antibiotics were tested in a competitive assay using homologous and heterologous conjugates coupled by various conjugation procedures as the ELISA solid phase. In contrast to the homologous assay recognizing only neomycin, the heterologous assay could be used for the detection of the whole subclass of deoxystreptamin antibiotics in buffer and raw milk. Gentamicin, kanamycin, and neomcyin were detected in artificially contaminated undiluted raw milk (without sample pretreatment) with 50% inhibition levels at 9, 21, and 113 ng mL(-)(1), respectively. Neomycin levels were also measured in milk samples obtained from a cow suffering from mastitis and treated with an antibiotic cocktail including neomycin. Levels below the EU maximum residue levels for deoxystreptamin antibiotics could readily be measured in this generic ELISA.     

Development of a Field Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of α-Amylase in Preharvest-Sprouted Wheat

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for detection of α-amylase in preharvest sprouted wheat and adapted to rapid field-use formats requiring 15–20 min to perform. Polyclonal and monoclonal antibodies were prepared to detect a mixture of high and low pI isozymes of α-amylase and high pI isozymes only. All antibodies detected α-amylase on immunoblots of either a crude wheat extract or of purified enzyme, but only the polyclonal antibodies functioned in a sandwich ELISA. Depending on the antibody combination, the tube ELISA detected either the high and low pI isozymes of α-amylase or the high pI isozymes only with a detection limit of ≈0.5–1.0 ng/mL of amylase. Wheats with falling numbers (FN) of <350 sec could be discriminated from sound wheats, with decreasing FN producing increasing assay color. Using 130 wheat grain samples, ELISA absorbances for detection of both high and low pI isozymes and of high pI isozymes only were highly positively correlated with amylase enzyme activity and negatively correlated with FN. The correlations were similar for detection of both isozyme families and for detection of high pI isozymes only. Analyses of three sets of wheat samples from different environments demonstrated that the relationship between ELISA absorbance and FN had little dependence on wheat cultivar. The precision of sample analysis using the field ELISA was similar to the precision of FN test apparatus.     

Elisa to quantify hexanal-protein adducts in a meat model system.

Monoclonal antibodies (MAb) were produced to hexanal-bovine serum albumin conjugates. An indirect competitive ELISA was developed with a detection range of 1-50 ng of hexanal/mL. Hexanal conjugated to three different proteins was recognized, whereas free hexanal and the native proteins were not detected. The antibody cross-reacted with pentanal, heptanal, and 2-trans-hexenal conjugated to chicken serum albumin (CSA) with cross-reactivities of 37.9, 76.6, and 45.0%, respectively. There was no cross-reactivity with propanal, butanal, octanal, and nonanal conjugated to CSA. The hexanal content of a meat model system was determined using MAb and polyclonal antibody-based ELISAs and compared with analysis by a dynamic headspace gas chromatographic (HS-GC) method and a thiobarbituric acid reactive substances (TBARS) assay. Both ELISAs showed strong correlations with the HS-GC and TBARS methods. ELISAs may be a fast and simple alternative to GC for monitoring lipid oxidation in meat.     

Synthesis of haptens and development of an immunoassay for the olive fruit fly pheromone

An enzyme-linked immunosorbent assay (ELISA) for the olive fruit fly pheromone, Bactrocera oleae Gmelin, was developed. The assay uses polyclonal antibodies, raised in rabbits, against (+/-)-beta-[3-(1,7-dioxaspiro[5.5]undecane)]propionic acid, 2 (hapten I), conjugated to the KLH (keyhole limpet hemocyanin) by the carbodiimide method. A second hapten, (+/-)-delta-[3-(1,7-dioxaspiro[5.5]undecane)]butylamine, 3 (hapten II), after conjugation to a biotin moiety, was used for indirect immobilization onto ELISA microwells precoated with the glycoprotein avidin. The developed ELISA method measures the synthetic olive fruit fly pheromone in concentrations ranging between 0.08 and 10 microg/mL and shows great promise for practical applications for pheromone detection in environmental and biological samples. The results obtained strongly indicate that this technique, to our knowledge the first insect pheromone enzyme-linked immunosorbent assay so far reported, is a fast, sensitive, inexpensive, and highly convenient method for the analysis of a volatile and low molecular weight compound such as 1,7-dioxaspiro[5.5]undecane, 1.     

Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody

Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.     

Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate

This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.