Comparative design of artificial ochratoxin A antigens: conjugation strategies and immunogenicity evaluation for antibody production.

Ochratoxin A (OTA) is a common fungal toxin contaminant in feed that is primarily produced by fungi belonging to the Aspergillus and Penicillium genera. In animal husbandry, the long-term ingestion of OTA-contaminated feed by animals can lead to severe consequences, such as reduced production performance, immunosuppression, and kidney and liver damage. Therefore, the development of efficient OTA detection technology is crucial for ensuring animal health and enhancing farming efficiency. This study aimed to design and modify the molecular structure of OTA, compare the preparation and identification of two artificial antigens, and select the optimal artificial antigen. Based on the molecular structure of OTA, the OTA hapten was activated via the N-hydroxysuccinimide active ester (NHS) method to generate an intermediate product known as OTA-carboxylic acid ester. This intermediate was subsequently conjugated with bovine serum albumin (BSA) via the coupling agents N, N'-dicyclohexylcarbodiimide (DCC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to prepare new and effective artificial antigens. Additionally, ovalbumin (OVA) was used to prepare detection antigens. The structures of the prepared artificial antigens were physically and chemically characterized via instrumental analysis techniques such as ultraviolet (UV) and infrared (IR) spectroscopy. Mice were immunized with artificial antigens to explore their immunogenicity. The results revealed successful conjugation of both artificial antigens with molecular binding ratios of OTA to BSA of 10.88:1 and 7.84:1. The immunized mice exhibited corresponding responses, and the titers of the mouse polyclonal antisera (pAbs) reached 1:3.2 × 103, the OTA pAb that was prepared from two artificial antigens was able to recognize OTA with 100% accuracy. Among them, the mice immunized with the artificial antigen OTA-BSA (EDC) exhibited the best sensitivity, with a half-maximal inhibitory concentration (IC50) of 11.40 ng/mL. Although the cross-reactivity rates with ochratoxin B (OTB) and ochratoxin C (OTC) were relatively high at 86.69% and 62.02%, respectively, the cross-reactivity rates with other compounds were less than 0.3%. In conclusion, this study successfully prepared two artificial antigens and selected the OTA-BSA (EDC) group as the optimal artificial antigen via identification. These results provide the antigenic foundation for the preparation of OTA monoclonal antibodies (mAbs) and the establishment of immunological analysis methods.