Application of a monoclonal-based immunoassay for the determination of imazalil in fruit juices

Abstract

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of imazalil [(RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole] in apple, tomato and orange juice samples. From an imazalil hapten, which mimics the analyte structure, several monoclonal antibodies were obtained. An ELISA in the conjugate-coated format was developed and optimized using the antibody showing the highest sensitivity. For standards, the detection limit of the ELISA was 0.2 nM (0.06 ng ml(-1)), with an I(50) value of 1.6 nM (0.5 ng ml(-1)). The study of the influence of matrices on assay reliability indicated that the ELISA could determine imazalil in fruit juices at the low ng ml(-1) level simply by diluting the sample, without any clean-up or concentration step. Recovery and precision of the method were evaluated by spiking juice samples with imazalil in the 10-500 ng ml(-1) range. The mean recovery from fruit juices was 97% and the mean coefficient of variation was approximately 20%. In addition to being precise and accurate, the method has proved to be simple and sensitive, with a quantification limit well below the maximum residue limits for imazalil in these matrices.

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

“…Immunoassays were very specific for imazalil, because the assayed fungicides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 (climbazole, hexaconazole, myclobutanil penconazole, prochloraz, propiconazole, tebuconazole, tetraconazole, and triadimefon) were not recognized (cross-reactivity <0.1 %) (Moreno et al 2007). With regard to the specificity of thiabendazole antibodies, the cross-reactivity pattern of RAbs was found to be very similar to that of the parent monoclonal antibody ( Table 2).…”

“…The production of the parent monoclonal antibodies TN3C-33, IMH-51, and DTPH-41 (raised against thiabendazole, imazalil, and tetraconazole, respectively) has been previously described (Abad et al 2001;Manclús et al 2008;Moreno et al 2007). For each hybridoma, 5×10 6 cells were pelleted by centrifugation at 780g for 5 min and resuspended in 400 µl of mRNA extraction buffer.…”

“…MAb-based ELISAs were performed using specific hapten coating conjugates as previously described (Abad et al 2001;Manclús et al 2008;Moreno et al 2007). RAbbased ELISAs were performed as follows.…”

“…Therefore, a round of panning was performed searching for functional imazalil-specific scFvs. Panning was performed in ELISA plate wells coated with 10 µg/ml of OVA-IMH (Moreno et al 2007). After the first round of panning, 6 of 96 randomly selected colonies rendered wells with scFv-pIII antibodies recognizing imazalil.…”

Study of the gastrointestinal biotransformation of zearalenone in a Caco‐2 cell culture system with liquid chromatographic methods

“…Due to its application, IMA residues can remain in fruits and obviously in the derived products. In the EU, the current legislation allows IMA residue levels up to 5 mg kg −1 in fresh fruit (Moreno et al, 2007). IMA has a strong inducing effect on CYP 1A1, an isoform of cytochrome P450 (CYP450; Sergent et al, 2007).…”