Shiro Miyake

Review of Enzyme-Linked Immunosorbent Assays (ELISAs) for Analyses of Neonicotinoid Insecticides in Agro-environments

Immunoassay is a promising method that is suitable for rapid and simple analyses of pesticides, which are likely to persist at a trace level in agro-environments, including agricultural products, soil, and water. Particularly, enzyme-linked immunosorbent assay (ELISA) has wide application to development of analytical methods for pesticide residues because it can very sensitively and very accurately determine them in samples. This paper presents a review of the fundamental analytical performance, a device for the sample pretreatment methods before determination, and cases of applications to various samples on ELISA methods that have been developed for detection of neonicotinoid insecticides in food or environmental matrices. The reviewed ELISAs can be ranked as quantitative, rapid and simple analytical methods for single analytes. The recognition of ELISA as an analytical methodology for pesticide residues is expected to advance rapidly in the future.

Analytical evaluation of enzyme-linked immunosorbent assay for neonicotinoid dinotefuran for potential application to quick and simple screening method in rice samples.

The analytical performance of a kit-based enzyme-linked immunosorbent assay (ELISA) for the determination of a neonicotinoid insecticide dinotefuran residue in rice samples is addressed. The sensitivity (I(50) value) was 5.4 ng/mL, with the limit of detection, 0.6 ng/mL and the dynamic range from 1.0 to 30 ng/mL. The ELISA showed substantially high specificity toward dinotefuran besides clothianidin (184%). For rice samples, dinotefuran was extracted with methanol and the extracts were directly determined with the ELISA because of no significant matrix interference. Good recoveries were observed and ranged from 92.5% to 113.2% with coefficients of variation below 10%. The results obtained with the ELISA correlated well with those by the HPLC method for rice samples (r>0.98). These findings strongly indicate that the evaluated and validated ELISA has a potential utility in a quick, simple, and reliable residue analysis, especially a screening method before shipment contributing to food safety.

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 (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 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.

Polyclonal and monoclonal antibodies specific to the chrysanthemic acid moiety of pyrethroid insecticides

Two polyclonal and two monoclonal antibodies raised against chrysanthemic acid (CAA) were prepared for ELISA of pyrethroid insecticides with the common CAA moiety. The monoclonal antibody KCA226 showed the highest reactivity towards allethrin among the antibodies in C-ELISA, although KCA226 was more sensitive to higher concentrations of methanol than the polyclonal antibodies in ELISA. KCA226 was reacted linearly with allethrin at concentrations ranging from 1 to 10 μg litre-1 in 0·5% methanol and from 300 to 10000 μg litre-1 in 30% methanol. KCA226 reacted specifically with the pyrethroid insecticides with the CAA moiety but was much less reactive with CAA itself. These results suggest that C-ELISA based on KCA226 is useful for the assay of pyrethroid residues with the common CAA moiety.

A scFv Antibody-Based Immunoaffinity Chromatography Column for Clean-Up of Bisphenol A-Contaminated Water Samples

This is a report on the development of immunoaffinity chromatography using a column of silica gel with an immobilized single-chain variable fragment (scFv) antibody specific to bisphenol A (BPA) for cleanup of BPA-contaminated water samples. The BBA-2187 scFv antibody specific to BPA was purified from the periplasmic fractions of the recombinant Escherichia coli. After a sample of BPA-contaminated river water was applied to the immunoaffinity column, the background signal intensity observed in high-performance liquid chromatography (HPLC) analysis of the eluates was markedly lower than that observed in HPLC analysis of the eluates from an Oasis HLB cartridge treated with the same sample. The immunoaffinity column efficiently concentrated BPA from actual river water samples with different matrices. Our results demonstrate that the immunoaffinity column with immobilized BBA-2187 scFv antibody is efficient for the cleanup of BPA-contaminated water samples from different sources.

Analysis of the Fungicide Boscalid in Horticultural Crops Using an Enzyme-Linked Immunosorbent Assay and an Immunosensor Based on Surface Plasmon Resonance

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an immunosensor based on surface plasmon resonance (SPR-sensor) were developed for fungicide boscalid determination in horticultural crops. To produce antiboscalid monoclonal antibodies (MoAb BSC7 and MoAb BSC72) for these assays, a hapten of boscalid was synthesized and conjugated to keyhole limpet hemocyanin for Balb/c mouse immunization. The working range of the dc-ELISA was 0.8-16 ng/mL with MoAb BSC7 and 2.5-120 ng/mL with MoAb BSC72, and that of the SPR-sensor was 17-80 ng/mL with MoAb BSC7. The dc-ELISA and SPR-sensor were compared for their sensitivity in determining boscalid residues at the maximum residue limit of 1-40 mg/kg for horticultural crops in Japan. Recovery of the spiked boscalid was 85-109% by the SPR-sensor and 100-124% by the dc-ELISA. On real tomato samples, the results obtained by both of these immunoassays correlated well with the results obtained by high-performance liquid chromatography.

Development of an Immunosensor for Determination of the Fungicide Chlorothalonil in Vegetables, Using Surface Plasmon Resonance

An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.

Immunochemical approach for assay of herbicide thiobencarb

Competitive enzyme-labeled immunosorbent assays (C-ELISAs) are simpler assays than instrumental analyses. Therefore, we attempted to develop a C-ELISA for assaying thiobencarb. The monoclonal antibody TBC 7-2 we produced was highly reactive with thiobencarb in this C-ELISA. Using the C-ELISA, thiobencarb-spiked water and crop samples were assayed.