Submitted to: Mycotoxin Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2017
Publication Date: 4/5/2017
Citation: Maragos, C.M., Sieve, K.K., Bobell, J. 2017. Detection of cyclopiazonic acid (CPA) in maize by immunoassay. Mycotoxin Research. 33(2):157-165.
Interpretive Summary: Cyclopiazonic acid (CPA) is a naturally occurring neurotoxin that is produced by certain fungi, in particular the species Aspergillus flavus. Many strains of A. flavus also produce the more widely known aflatoxins, and these two groups of toxins are known to occur together in commodities. Despite this, immunoassays have not been widely developed to screen for CPA in commodities or foods. To address this issue, antibodies and associated immunoassays were developed at the Mycotoxin Prevention and Applied Microbiology Research Unit (Peoria, IL). In particular, two monoclonal antibodies were developed and used to produce very sensitive enzyme-linked immunosorbent assays (ELISAs). One of the antibodies was further used to develop an assay for measuring CPA in maize over the range from 5 to 28 µg/kg (ppb). The ELISA tended to overestimate CPA content, a difference believed to be due to the presence of related "masked" derivatives of CPA. The antibodies developed and the resulting screening assay will be useful tools for the further evaluation of the prevalence of this mycotoxin in maize.
Technical Abstract: Cyclopiazonic acid (a-CPA) is a tremorgenic mycotoxin that is commonly produced by certain of the Aspergilli, in particular A. flavus, which is more widely known for production of the aflatoxins. Despite the fact that a-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for a-CPA. Two mAbs in particular were very sensitive, with IC50s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC20 to IC80) was from 5 to 28 µg/kg. Recoveries from maize spiked over the range from 2 to 50 µg/kg averaged 88.6 ± 12.6%. Twenty eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the 5 samples above the limits of quantitation of both methods, the CI-ELISA tended to overestimate CPA content, a difference we speculate may be due to related metabolites or perhaps “masked” derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for the further evaluation of the prevalence of this mycotoxin in maize.