A label-free, direct solid-phase fluorimetric analysis of ochratoxin A in agricultural products with monoclonal antibody-immobilized monolith.

We established a method for directly measuring mycotoxin ochratoxin A (OTA) in foods by solid-phase fluorescence of monolith-immobilized antibodies. The antibody was introduced onto only one side of an 8 mm-diameter, 3 mm-thick monolith via covalently immobilized protein G. 4 µg (2.7 × 10-11 mol) of antibody was immobilized per one monolith. A maximum of 10 µg (2.4 × 10-11 mol) OTA adsorbed to the activated side of each monolith. The amount of OTA adsorbed and the fluorescence intensity showed good linearity in the range of 0.5-3 ng OTA. Loading the sample solution onto the non-antibody side on the monolith blocked the hydrophobic fluorescent matrices from reaching the immobilized surface of the antibody. The proposed method was able to detect 1 ng OTA/g in solid samples with complex matrices. Mean recoveries obtained at spiked concentration of 3 ng g-1 OTA/g were 78-90% with relative standard deviations of <7.9%.

Development of immunoassay based on monoclonal antibody reacted with the neonicotinoid insecticides clothianidin and dinotefuran.

Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

Development of an enzyme-linked immunosorbent assay (ELISA) for residue analysis of the fungicide azoxystrobin in agricultural products.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for residue analysis of azoxystrobin in garden crops, for which the maximum residue limits (MRLs) are 0.5-50 mg/kg in Japan. For hapten synthesis, an ethyl carboxyl group was introduced to the 4-position of the 2-cyanophenoxy group in azoxystrobin, and its cyano group was changed to a methyl group. An anti-azoxystrobin monoclonal antibody was prepared from mice immunized with hapten-keyhole limpet hemocyanin conjugate. The dc-ELISA using prepared antibody showed 50-250-fold higher sensitivity compared to the MRLs. The working range of the dc-ELISA was 10-200 ng/mL. The dc-ELISA showed high specificity to azoxystrobin. When methanol extracts from nine kinds of garden crops spiked with azoxystrobin ranging near the MRLs were analyzed, the determined results by the dc-ELISA agreed well with the results of their controls. In addition, azoxystrobin spiked in garden crops homogenates was satisfactorily extracted by methanol solution and easily analyzed. The recovery rate of dc-ELISA was 96-109% and correlated well with the results obtained by HPLC analysis.

Development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody.

Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.

Development of a novel immunoaffinity column for aflatoxin analysis using an organic solvent-tolerant monoclonal antibody.

An organic solvent-tolerant monoclonal antibody specific to aflatoxins B(1), B(2), G(1), G(2), and M(1) (AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1)) was prepared. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) values were 1.9, 2.1, 2.1, 2.4, and 2.8 ng/mL for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1), respectively. Antibody reactivity was retained at 40% methanol concentration or at acetonitrile concentrations up to 40%. An immunoaffinity column (IAC) was prepared using agarose gel beads with bound antibody. The IAC retained the tested AFs that were 89, 90, 95, 90, and 89% for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1) at 20% acetonitrile concentrations or that were 81, 87, 79, and 83% for AFB(1), AFB(2), AFG(1), and AFG(2) at 60% methanol concentrations. Roasted peanuts and seven kinds of spices were spiked with 8.0, 1.0, 6.0, and 1.0 ng for AFB(1), AFB(2), AFG(1), and AFG(2) per 1 g sample and extracted with 90% acetonitrile. The roasted peanuts and cayenne pepper out of the spices were also extracted with 70% methanol. The extracts were diluted 5-fold with phosphate-buffered saline and applied to the IAC. The spiked aflatoxins were recovered with satisfactory rates: 78 (RSD, 2.1%) to 127% (RSD, 1.7%). The developed IAC was used for the analysis of aflatoxins in naturally contaminated samples of roasted peanuts and cayenne pepper. The newly developed IAC showed substantially organic solvent tolerance at the concentration that could not be used for existing IACs, and the column showed good ability to clean up samples for food analysis.

Development of an enzyme-linked immunosorbent assay for residue analysis of the insecticide emamectin benzoate in agricultural products.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the analysis of emamectin residues in agricultural products was developed using a prepared mouse monoclonal antibody. The working range was 0.3-3.0 ng/mL, and the 50% inhibition concentration (IC(50)) was 1.0 ng/mL. The assay was sufficiently sensitive for analysis of the maximum residue limits in agricultural products in Japan (>0.1 microg/g). Emamectin residues contain the following metabolites: the 4''-epi-amino analogue, the 4''-epi-(N-formyl)amino analogue, the 4''-epi-(N-formyl-N-methyl)amino analogue, and the 8,9-Z isomer. The dc-ELISA reacted with these compounds at ratios of 113, 55, 38, and 9.1% of the IC(50) value of emamectin benzoate. Seven kinds of vegetables were spiked with emamectin benzoate at concentrations of 15-300 ng/g, and the recoveries were 91-117% in the dc-ELISA. The dc-ELISA results agreed reasonably well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using spiked samples and actual (incurred) samples. The results indicate that the dc-ELISA was useful for the analysis of emamectin benzoate residues in agricultural products.