**Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin**

#### **Chris M. Maragos 1,\*, Yosuke Uchiyama 2, Naoki Kobayashi 2, Fumichika Kominato 3 and Yoshiko Sugita-Konishi 2**



Received: 10 October 2019; Accepted: 28 October 2019; Published: 30 October 2019

**Abstract:** 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 di fficult 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 sugges<sup>t</sup> 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.

#### **Keywords:** citreoviridin; antibody; immunoassay; rice

**Key Contribution:** Two monoclonal antibodies were produced and used to develop a screening immunoassay for detecting the mycotoxin citreoviridin at relevant levels in white rice.
