Determination of Severe Peach Allergens, Gibberellin-Regulated Protein, and Lipid Transfer Protein, Using Monoclonal Antibodies.

In this study, monoclonal antibodies against two major fruit allergens-gibberellin-regulated protein (GRP) and lipid transfer protein (LTP)-were established. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantification of peach GRP and LTP were constructed using these antibodies. Both ELISAs reacted with the respective antigens when heated at 100ºC for 20 min, but not when reduced with sodium sulfite, indicating that GRP and LTP are heat-stable, while disulfide bonds play an important role in their native steric structures. GRP and LTP in peaches and peach-containing foods were quantified by these ELISAs. In both cases, there were few differences among peach cultivars normally available on the market; however, concentrations were higher when the peach was ripe. GRP was localized in the pulp of the peach, while LTP was present in the peel. They could be quantified in peach-containing beverages, as well as in dried and canned peaches. GRP in Japanese apricots could also be determined using this ELISA, as its amino acid sequence is the same as that of peach GRP. Then, high concentrations of GRP were detected in umeboshi, a traditional Japanese pickled apricot. Peach leaves were found to have a high LTP content, accordingly, LTP was also observed in lotions containing peach leaf extract. The ability to quantitatively detect GRP and LTP in this study will, therefore, contribute to the improvement of component-resolved diagnoses and quality of life in patients allergic to peaches.

Development of direct competitive ELISA for residue analysis of fungicide chlorothalonil in vegetables.

A direct competitive (dc)-ELISA was developed for rapid and simple determination of chlorothalonil residue in vegetables. A carboxylic acid derivative of pentachlorophenol was used to prepare an anti-chlorothalonil monoclonal antibody (MoAb) that showed adequate reactivity for dc-ELISA. Before homogenization of vegetable samples, phosphoric acid was added (vegetable-10% phosphoric acid (2 : 1, w/v)) to block enzymatic decomposition of chlorothalonil. The use of phosphate buffer (100 mmol/L, pH 7.0) minimized the influence of phosphoric acid on competitive reaction in the dc-ELISA. Working range was 0.10 to 6.0 ng/mL in the optimized dc-ELISA. The recovery of chlorothalonil spiked in cucumber and eggplant was 97.1 to 125%. The results correlated well with those obtained by HPLC analysis. The dc-ELISA could rapidly determine chlorothalonil after a simple sample preparation procedure.

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.