Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Residue Analysis of the Fungicide Azoxystrobin in Agricultural Products

Abstract

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 and Application of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of Amygdalin, a Cyanogenic Glycoside, in Food

“…However, high levels of amygdalin were detected in fruit seeds 13 (0.04 -4.52 mg g -1 ) and kernels (2.14 -9.75 mg g -1 ). Lower levels of amygdalin 14 observed in juices and processed product could be as a result of the effect of In this study, a highly specific polyclonal antibody was raised against amygdalin 9 using a hapten linked to a carrier protein through a very simple conjugation technique.…”

“…5,6,7,8,9 8 Several important plant crops, such as cassava, sorghum, millet, barley, almond, 9 cherry, macadamia nut, peach, lima beans, and kidney beans, have been reported to 10 biosynthesize and accumulate cyanogenic glycosides. 1,10,11,12 Traditional and modern 11 food processing techniques such as chopping, grinding, soaking, fermentation, drying, 12 roasting, boiling, and steaming have been used to reduce or eliminate the potential 13 toxicity of dietary cyanogenic plants. 13 These methods can be effective because HCN equivalents for canned stone fruits.…”

“…1,10,11,12 Traditional and modern 11 food processing techniques such as chopping, grinding, soaking, fermentation, drying, 12 roasting, boiling, and steaming have been used to reduce or eliminate the potential 13 toxicity of dietary cyanogenic plants. 13 These methods can be effective because HCN equivalents for canned stone fruits.…”

“…Secondly, the trinitrobenzene sulfonic acid assay (TNBS; Habeeb 25 ) was used to 13 estimate the molar conjugation ratio of amygdalin-BSA and amygdalin-KLH conjugates. Immunoassays are now widely used in quantitative analysis for agric-food 8 components for their advantages of cost-effectiveness and simplicity which arise directly 9 from the affinity of the antibody interaction with its target.…”

“…Surprisingly, perhaps, given 10 that immunoassays can be used away from fully-equipped laboratories to monitor food 11 processing, only 2 previous reports have described antibody production against 12 cyanogenic glycosides, both against amygdalin. 22,23 No antibody characterization was 13 carried out, no assay validation reported and nor was any quantitative data provided.…”