Preparation and Characterization of Ochratoxin A–Bovine Serum Albumin Conjugate Using EDC/NHS Chemistry for Immunodiagnostic Applications

Background: Ochratoxin A (OTA) is an important mycotoxin produced by Aspergillus and Penicillium species of fungi. It is a potent nephrotoxic and carcinogenic mycotoxin affecting poultry and other animals, including humans. All mycotoxins are low-molecular-weight proteins, and for their detection, they must be conjugated with bovine serum albumin (BSA). This conjugation makes them immunogenic and allows them to be used as coating antigens in immunological assays. Aims: This study aimed to conjugate OTA with BSA by activating the free carboxyl group of OTA with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), followed by coupling to BSA amino groups. Place and Duration of Study: This study was conducted at the Department of Pathology, Mamnoor, Warangal, from January 2026 to September 2026. Methodology: OTA (5 mg) was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO), mixed with NHS (6 mg) and EDC (8 mg), and incubated for 2 h 30 min at room temperature in the dark, followed by overnight incubation at 4°C. The activated OTA was added dropwise to BSA (10 mg) in carbonate buffer, pH 9.6, and stirred for 2 h 30 min. The mixture was dialysed against PBS for 72 h to remove free OTA and reagents. Results: OTA was successfully activated using EDC and NHS and coupled to BSA through its free carboxyl group. The reaction mixture was purified by extensive dialysis, and a clear OTA–BSA conjugate solution was obtained. SDS-PAGE analysis showed a major band corresponding to native BSA at approximately 66 kDa. The OTA–BSA conjugate also showed a band in the BSA region, but with a slight upward shift in apparent molecular weight compared with native BSA. This shift indicated that OTA molecules were covalently attached to BSA. Conclusion: These findings indicate successful conjugation of OTA to BSA. The OTA–BSA conjugate can be used as an immunogen or coating antigen for the development of OTA-specific immunoassays.