The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA.

Citrinin (CTN) is a hepato-nephrotoxic mycotoxin produced by fungi genera of Aspergillus, Monauscus, and Penicillium. CTN contaminates grains, fruits, juices and vegetables, and causes various toxic effects to humans and animals. It has small molecular weight, which is non-immunogenic to animals. Thus, CTN was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, by amide bonds using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Mice were immunized with CTN-BSA conjugates, and spleen cells of the immunized mice were fused with Sp2/0 myeloma cells to obtain 21H27 hybriodoma cell. Ascitic fluid of hybridoma cell was produced in mice abdomen, and purified using caprylic/ammonium sulfate precipitation method. The 21H27 anti-CTN mcAb was the IgG2a antibody subclass, and cross-reactivity results indicated that anti-CTN mcAb is specific to CTN with high affinity (2.0 × 108 L/mol). Indirect competitive ELISA (ic-ELISA) results showed that the linear range of detection was 0.01-5.96 ng/mL and the IC50 was 0.28 ng/mL with a lower detection limit (LOD) of 0.01 ng/mL. The average recovery was 93.8% ± 1.6% with a coefficient variation of 1.0%-4.3%. Hence, anti-CTN mcAb secreted by 21H27 hybridoma cell was successfully produced and can be used to detect CTN contaminated feed and foodstuffs.

The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence.

DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.