Determination of ochratoxin A by polyclonal antibodies based sensitive time-resolved fluoroimmunoassay

Abstract

Using indirect competitive time-resolved fluoroimmunoassay (TRFIA), a rapid, highly selective and extremely sensitive method has been established for the determination of ochratoxin A (OA). Tests can be performed in a 96-well microplate using the toxin-specific polyclonal antibodies, obtained from rabbits immunized with ochratoxin A-keyhole limpet's hemocyanin (OA-KLH). In indirect TRFIA format, ochratoxin A-bovine serum albumin conjugate (OA-BSA) is coated onto the microtitre plate and incubated with standard toxin (samples) and anti-OA antibody. A goat anti-rabbit IgG Eu(3+) conjugate is used to enable the detection. The suitability of the assay for quantification of OA is also studied and samples are determined by OA-TRFIA using autoDELFIA1235 system. The results show that the polyclonal antibodies can be used at a dilution exceeding 1:8,000 and the OA detection limit is 0.02 microg/l for indirect competitive TRFIA formats. The 80, 50, and 20% inhibition binding effect dose (ED80, ED50, ED20) of OA were 0.195, 1.018, and 5.314 microg/l, respectively. The assay ranges from 0.02 to 400 microg/l. The cross reactivity with ochratoxin B is 5.6% and antibodies do not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OA-TRFIA are 2.6 and 5.2%, respectively. The mean recoveries from the OA-free cereals spiked with 1-200 microg of OA/kg of cereals sample were 95.8%. Both OA-TRFIA and OA-ELISA tests are applied for the quantitative measurement of OA in the same cereals, and the coefficient of correlation is 0.912. The results show that the novel TRFIA method can be applied to detect the OA contamination in cereals. It provides very high sensitivity and optimal range and will be useful to screen OA contamination easily, simply, and economically when the number of samples is large.

Development of a sensitive competitive indirect ELISA method for determination of ochratoxin A levels in cereals originating from Nanjing, China

“…The CR of anti-OTA mAb for OTB was 17% with an IC 50 of 10 ng/mL, which was lower than in some previous studies, such as 31.7% (Cho et al, 2005), 100% (Clarke et al, 1993) and 98.8% (Kawamura et al, 1989), and was higher than in others, such as 9.3% (GyongyosiHorvath et al, 1996), 5.6% (Huang et al, 2006), 0.8% (Yu et al, 2005), and 1.07% (Fujii et al, 2006). The structure of OTA only differs from OTB at the C5 of the isocoumarin moiety of ochratoxin, which results in the CR of anti-OTA antibody with OTB.…”

“…Native contaminated samples (n ¼ 65) were collected from the Nanjing markets. The samples, either natural cereal samples or spiked cereal samples, were prepared as reported by Huang, Tao, Shi, Tang, and Jin (2006), with some modification. Briefly, 25 g cereal samples were crushed by household grinder and passed through a 20-mesh sieve; subsequently extracted with 100 mL 50% methanol in PBS for 30 min at room temperature, with shaking at intervals, centrifuged at 2000 rpm for 10 min and filtered through Whatman no.…”

Effects of different mycotoxins on humans, cell genome and their involvement in cancer

“…The most important ochratoxin is the Ochratoxin A, which is produced mainly by Aspergillus Ochraceus (62) and its chemical formula is C 20 h 18 C l NO 6 ( fig. 5) (63).…”

Future Applications of Electronic-Nose Technologies in Healthcare and Biomedicine

“…Other examples include the occurrence of mycotoxins, toxic secondary metabolites (e.g. aflatoxin and ochratoxin A) produced by fungi such as Aspergillus and Fusarium species that commonly grow on agricultural products in the field or in storage (Huang et al, 2006). Cheli et al (2009) very effectively utilized the PEN2 e-nose with principal component analysis (PCA) to detect the presence of aflatoxin in maize samples at a high level of confidence.…”