Presentation Information
[1ENZ-08]Development of scFv-alkaline phosphatase fusion protein for application to sensitive enzyme immunoassays
○Ngoc Minh Nguyen1, Miyu Omura1, Yoichi Kumada1 (1. Kyoto Inst. Technol. (Japan))
Keywords:
Alkaline phosphatase,Single-chain variable fragment,Fusion protein,Immunoassay
Enzyme immunoassays (EIAs) are traditional analytical methods that are based on the antigen-antibody interaction to detect target substances specifically, often visualized through colorimetric or chemiluminescent signals using enzyme-labeled antibodies. Especially, calf intestinal alkaline phosphatase (CIAP) is one of the popular enzymes for labeling with antibodies, and CIAP-labeled antibodies have been exclusively utilized in ELISA, Western blots, CLEIAs, and immunohistochemistry. On the other hand, the major concern was the reduction in specific activity, solubility and productivity of CIAP-labeled antibodies due to their laborious preparation process and a number of by-products from random cross-linking reactions between the antibody and CIAP.
The aim of the study was to design, produce, and characterize scFv-AP fusion proteins that can be prepared through simple cultivation and purification processes. Among 4 different APs tested, APs from human placental (HPAP) and Shewanella sp. T3-3 (T3AP) were effectively expressed in ExpiCHO cells even as scFv-AP forms. Both antigen-binding activities and catalytic activities could be highly maintained even after genetic fusion between scFv and APs. The scFv-AP fusion proteins could detect the target antigen with considerably higher signals in sandwich-type ELISA, CLEIA, and LFIA. In particular, the detection limit of antigen for scFv-T3AP was 100-fold lower than that for the conventional CIAP-labeled antibody in sandwich ELISA. Furthermore, the scFv-T3APs with different antigen-binding specificities or different thermal stabilities were successfully prepared by the original CDR-grafting technology. Thus, the highly functional scFv-T3AP fusion proteins developed in this study offer a promising tool for versatile application in enzyme immunoassays and would be expected as a next-generation of custom-made secondary antibody in a variety of EIAs.
The aim of the study was to design, produce, and characterize scFv-AP fusion proteins that can be prepared through simple cultivation and purification processes. Among 4 different APs tested, APs from human placental (HPAP) and Shewanella sp. T3-3 (T3AP) were effectively expressed in ExpiCHO cells even as scFv-AP forms. Both antigen-binding activities and catalytic activities could be highly maintained even after genetic fusion between scFv and APs. The scFv-AP fusion proteins could detect the target antigen with considerably higher signals in sandwich-type ELISA, CLEIA, and LFIA. In particular, the detection limit of antigen for scFv-T3AP was 100-fold lower than that for the conventional CIAP-labeled antibody in sandwich ELISA. Furthermore, the scFv-T3APs with different antigen-binding specificities or different thermal stabilities were successfully prepared by the original CDR-grafting technology. Thus, the highly functional scFv-T3AP fusion proteins developed in this study offer a promising tool for versatile application in enzyme immunoassays and would be expected as a next-generation of custom-made secondary antibody in a variety of EIAs.
Comment
To browse or post comments, you must log in.Log in