Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 µg L(-1)) and an IC(50) of 3.2 nM (0.64 µg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

Development of competitive immunoassays to hydroxyl containing fungicide metabolites.

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 µg/L and a limit of detection (LOD) of 1.1 µg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 µg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 µg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.

Strip-based immunoassay for rapid detection of thiabendazole.

There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL(-1) of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08+/-0.03 ng mL(-1) with an IC(50) value of 0.60+/-0.08 ng mL(-1) and a linear working range of 0.11-4.13 ng mL(-1). Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05-5 mg L(-1). Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.

Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure.

A novel technique for patterning immobilized antibody layers based upon photolithography and oxygen plasma exposure has been developed. Mouse monoclonal antibodies specific for thiabendazole (a post-harvest fungicide and veterinary anthelmintic) were covalently linked through free amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Immobilized antibody layers were stabilized with sucrose, dehydrated, and stored refrigerated with desiccant. Photolithographic patterning was performed with a positive photoresist with modified bake temperatures and times, selective UV exposure with a contact mask, and aqueous alkaline solubilization of exposed resist. Exposed regions of immobilized antibody were then removed by exposure to a low power, radio frequency oxygen discharge. Residual resist was stripped with acetone. Successful patterning was demonstrated by challenging surfaces with goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate. Sucrose stabilization was necessary for antibody to undergo photoresist processing without loss of binding activity. Challenge with enzyme linked antigen of oxygen plasma exposed antibody layers demonstrated that plasma treatment completely neutralized antibody capture ability. Ellipsometry measurements of oxygen plasma exposed antibody layers indicated complete removal of immobilized antibodies. Fluorescent imaging demonstrated smallest line widths of 2-3 microns.

Sicca complex and cholangiostatic jaundice in two members of a family probably caused by thiabendazole.

An entire family (father, mother, and three daughters) were given thiabendazole because one of the children had acquired pinworm infestation. The mother and one daughter (non-infected) developed a sicca complex (keratoconjunctivitis sicca and xerostomia) accompanied by cholangiostatic jaundice. Sjögren's syndrome is an autoimmune disease and presents many immune mechanism aberrancies. An association between autoimmune liver disease and sicca complex has been reported. Labeled mitochondrial antibodies bound to the parotid duct have been noted in patients with autoimmune cholangiostatic jaundice and such antibodies may be similar to the antibody against salivary duct found in Sjögren's syndrome. It is suggested that in these two patients, thiabendazole may have acted as a hapten and by binding to the body protein induced the production of autoantibodies which may have acted against the biliary epithelium, the salivary duct epithelium, and the lacrimal gland ducts.