Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans.

Aurovertins are the structurally diverse polyketides that distribute widely in different fungal species. They feature a 2,6-dioxabicyclo[3.2.1]-octane ring in structure and exhibit the potential antitumor activity against breast cancer as F1-ATPase ß subunit inhibitor. In this study, we constructed the biosynthetic pathway of aurovertin in an Aspergillus nidulans host and obtained seven aurovertin-type compounds. Surprisingly, three new aurovertin geometric isomers were characterized. By introducing an inducible promoter xylP(p) in the pathway gene acyltransferase aurG, we can control the product ratios among different aurovertin compounds by adding glucose and/or inducer xylose. The yields of aurovertins could be increased up to about 20 times by adding a constitutive promoter gpdA(p) to transcription factor AurF, which indicates AurF's positive role in the biosynthesis of aurovertin. Taken together, our results provided not only an efficient way to generate bioactive fungal natural products but also realized the rational controlling their yields with designed promoters.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 µg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

Screening and Molecular Evolution of a Single Chain Variable Fragment Antibody (scFv) against Citreoviridin Toxin.

Citreoviridin (CIT), a small food-borne mycotoxin produced by Penicillium citreonigrum, is generally distributed in various cereal grains and farm crop products around the world and has caused cytotoxicity as an uncompetitive inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv) antibody that can detect the citreoviridin in samples is still not available; therefore, it is very urgent to prepare an antibody for CIT detection and therapy. In this study, an amplified and assembled scFv from hybridoma was used to construct the mutant phage library by error-prone PCR, generating a 2 × 108 capacity mutated phage display library. After six rounds of biopanning, the selected scFv-5A10 displayed higher affinity and specificity to CIT antigen, with an increased affinity of 13.25-fold (Kaff = 5.7 × 109 L/mol) compared to that of the original wild-type scFv. Two critical amino acids (P100 and T151) distributed in H-CDR3 and L-FR regions that were responsible for scFv-5A10 to CIT were found and verified by oligonucleotide-directed mutagenesis, and the resulting three mutants except for the mutant (P100K) lost binding activity significantly against CIT, as predicated. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect CIT was 25-562 ng/mL with IC50 at 120 ng/mL. The limit of detection was 14.7 ng/mL, and the recovery average was (90.612 ± 3.889)%. Hence, the expressed and purified anti-CIT MBP-linker-scFv can be used to detect CIT in corn and related samples.

Preparation and identification of monoclonal antibody against Citreoviridin and development of detection by Ic-ELISA.

Citreoviridin (CIT), a neurotoxic mycotoxin produced by Penicillium citreonigrum is generally detected in cereal grains and agricultural products worldwide, and has numerous toxicological effects on human and animal health. Therefore, it is necessary to develop a rapid, sensitive, and reliable immunoassay method for CIT. In this study, artificial antigen CIT-KLH and CIT-BSA was successfully prepared via succinic anhydride and carbodiimide two-step method. CIT-KLH conjugates were injected into Balb/c mice, and titer of the antiserum against CIT was determined using CIT-BSA as coating antigen by ELISA method. A hybridoma cell line 8D8 stably secreting monoclonal antibody against CIT was generated by fusing SP2/0 myeloma cells with the splenocytes from the immunized mice. The titer of 8D8 mAb reached 1: 1.28 × 10(5) after purified by caprylic/ammonium sulfate precipitation (CA-AS) method. The 8D8 mAb was identified as IgG1 subtype. The cross-reactivity results indicated that anti-CIT mAb was highly specific to Citreoviridin, and the average affinity 4.57 × 10(8) L/mol. A sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for CIT was established. Under optimal condition, the linear range to detect CIT was 11.02-2370.48 ng/mL with IC50 of 161.66 ng/mL and the limit of detection of the ic-ELISA was 11.86 ng/mL. With the mean coefficient of variation lowing 5%, the mean recovery in intra-assay and inter-assay were (90.06 ± 1.60)% and (89.65 ± 1.69)%, respectively. Therefore, the anti-CIT mAb secreted by 8D8 hybridoma cell line is useful for analysis of food contaminated with CIT.

Artificial antigen synthesis and the development of polyclonal antibody-based immunoassay for citreoviridin determination.

Citreoviridin, a mycotoxin produced by Penicillium citreonigrum is a common contaminant of wide range of agri-products and detrimental to human and animal health. Therefore it is important to develop a rapid, sensitive, and specific immunoassay for citreoviridin detection. In this study, polyclonal antibody against citreoviridin was developed. For the preparation of citreoviridin-bovine serum albumin conjugate (CIT-BSA), hydroxyl groups on adjacent carbon atoms were oxidized by sodium periodate, so the product with reactive aldehyde residues was suitable for coupling with amine. Anti-citreoviridin polyclonal antibody was prepared by immunizing mice with CIT-BSA conjugate. The specificity and sensitivity of the polyclonal antibody was determined by indirect competitive ELISA. Results showed that the IC50 value of the polyclonal antibody was 0.56 µg/mL and no cross-reactivity was found between antiserum and other mycotoxins used in the experiment. The citreoviridin recovery rates by this polyclonal antibody were calculated through rice powder spiked by artificial citreoviridin. The recovery rates ranged were found from 70.5 ± 0.08 % to 94.7 ± 0.09% for inter-assay, and from 77.5 ± 0.04% to 95.4 ± 0.18% for intra-assay, which indicated that this polyclonal antibody could detect trace amount of CIT from the tested samples. Consequently, this study provided a specific and sensitive anti-citreoviridin polyclonal antibody, which made the determination of citreoviridin easier, quicker, and more accurate.

Co-occurrence of aflatoxins B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, and citreoviridin in rice in Brazil.

A total of 230 samples of processed rice and its sub-products or derived products were analysed to establish the co-occurrence of several mycotoxins. Samples were analysed in the period 2007-2009 due to the outbreak of beriberi associated with the consumption of rice stored in inappropriate conditions in Brazil. According to data from the Ministry of Health, 323 cases of disease were registered in 2006, of which at least 47 cases resulted in death. The occurrence of total aflatoxin (AFT) (aflatoxin B(1) + B(2) + G(1) + G(2)), ochratoxin A (OTA), zearalenone (ZON), deoxynivalenol (DON), and citreoviridin (CTV) was 58.7%, 40.0%, 45.2%, 8.3% and 22.5%, respectively. From 166 rice samples analysed, 55% had levels <0.11 µg kg(-1) for AFT. For OTA and ZON, of 165 rice samples analysed, 28% and 29% were contaminated with levels from 0.20 to 0.24 µg kg(-1) and from 3.6 to 290.0 µg kg(-1), respectively. One sample (0.6%) was contaminated with 4872.0 µg kg(-1) of ZON. A total of 91% of rice samples (n = 165) did not contain detectable DON (<30.00 µg kg(-1)), although the highest level of contamination was found to be 244 µg kg(-1). From the total of 65 samples analysed, 94% had no detectable CTV (<0.9 µg kg(-1)), with a range from 0.9 to 31.1 µg kg(-1) in 6% of the samples. The highest levels of contamination were found in rice sub-products or derived products from the husk and rice bran. Co-occurrence was observed for AFT and ZON in 17.0%, AFT and OTA in 24.2%, AFT and CTV in 6.2%, OTA and CTV in 4.6%, and ZON and CTV in 3.1%. These fractions were also the major contributors for the co-occurrence. The results found show the necessity of monitoring rice production.

Liquid chromatographic method for determination of citreoviridin in corn and rice.

Citreoviridin, a neurotoxic mycotoxin, has been found as a natural contaminant in corn left unharvested in the southeastern United States and in rice of several Asian countries, including Japan. A reliable analytical method for the quantitative determination of citreoviridin in corn and rice is described. Corn or rice is extracted with dichloromethane, and the extract is partially purified on silica and amino solid-phase extraction (SPE) columns. The extract is analyzed for citreoviridin by normal-phase liquid chromatography, using a mobile phase of ethyl acetate-hexane (75 + 25) at 1.5 mL/min and a fluorescence detector to measure the yellow fluorescence (388 nm excitation, 480 nm emission). With a 100 microL injection loop, the relationship between concentration and injection volume is linear for 20-60 microL injections. Recoveries of citreoviridin added to yellow corn at 10-50 ng/g were 91.0-96.9%; recoveries from white corn (10-50 ng/g added) were 96.8-102.8%. Recoveries of 5000 ng/g added to white corn were 89.0%, indicating that heavily contaminated samples can be assayed by the method. Minimum detection limits were 10 ng for citreoviridin standard and 2 ng/g for citreoviridin added to corn. White rice fermented with Penicillium citreo-viride (1524 ppm) was mixed with and serially diluted with uncontaminated ground corn to obtain citreoviridin-contaminated corn (ca 25 ppb). When the samples were assayed by the method, a mean level of 24.4 +/- 1.65 ppb (6.5% coefficient of variation) was obtained. Four fermented rice food samples and 3 commercial rice samples were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Citreoviridin levels in Eupenicillium ochrosalmoneum-infested maize kernels at harvest.

Citreoviridin contents were measured in eight bulk samples of maize kernels collected from eight fields immediately following harvest in southern Georgia. Citreoviridin contamination in six of the bulk samples ranged from 19 to 2,790 micrograms/kg. In hand-picked samples the toxin was concentrated in a few kernels (pick-outs), the contents of which were stained a bright lemon yellow (range, 53,800 to 759,900 micrograms/kg). The citreoviridin-producing fungus Eupenicillium ochrosalmoneum Scott & Stolk was isolated from each of these pick-out kernels. Citreoviridin was not detected in bulk samples from two of the fields. Aflatoxins were also present in all of the bulk samples (total aflatoxin B1 and B2; range, 7 to 360 micrograms/kg), including those not containing citreoviridin. In Biotron-grown maize ears that were inoculated with E. ochrosalmoneum through a wound made with a toothpick, citreoviridin was concentrated primarily in the wounded and fungus-rotted kernels (range, 142,000 to 2,780,000 micrograms/kg). Samples of uninjured kernels immediately adjacent to the wounded kernel (first circle) had less than 4,000 micrograms of citreoviridin per kg, while the mean concentration of toxin in kernel samples representing the next row removed (second circle) and all remaining kernels from the ear was less than 45 micrograms/kg. Animal toxicosis has not been linked to citreoviridin-contaminated maize.