Application of two novel anionic peroxidases from Raphanus sativus L. var niger roots in labeling antibodies and developing an enzyme-linked immunosorbent assay

HRP, or horseradish peroxidase, is a reporter enzyme with extensive use in biotechnological applications. We previously reported the purification and characterization of two anionic peroxidases from <i>Raphanus sativus</i> L. var <i>niger</i> (black radish) roots. Here, we evaluated the applicability of these two novel peroxidases as alternatives to traditional horseradish peroxidase (HRP). The two novel peroxidases (BRP-A and BRP-B) and HRP were conjugated to IgY polyclonal antibodies by chemical methods based on the use of sodium periodate and cyanuric chloride. Moreover, the applicability of BRP-A and BRP-B in immunoassays was investigated by comparing the signal generated by these novel peroxidases in ELISA with HRP conjugates. Additionally, the limit of detection (LOD) was calculated for BRP-A, BRP-B, and HRP conjugates. Finally, the thermal stability of peroxidase antibody conjugates at 37 °C and 4 °C was compared. The peroxidase antibody conjugates prepared by the periodate method generated a much stronger signal than those prepared by the cyanuric chloride method. The signal obtained by BRP-A and BRP-B conjugates was much lower compared to the commercial HRP enzyme. The limit of detection was found to be 385.71, 213.75, and 43.6 ng per well for BRP-A, BRP-B, and HRP conjugates prepared by the periodate method, respectively. However, for conjugates prepared by the cyanuric chloride method, the limit of detection could only be estimated for HRP since BRP-A and BRP-B had an extremely low signal-to-noise ratio. All peroxidase conjugates had comparable thermal stability at 37 °C and 4 °C.