An efficient functional screening method for anti ATP hydrolase antibody based on fluorescent probes.

Antibody drugs as one of the important categories of drugs in the modern biomedicine filed have been successfully applied in various fields, such as cancer diseases and immune disorders. In the early stage of monoclonal antibody drug development, scientists need to accurately select the highly functional antibodies from the numerous candidates obtained from immunized animals for subsequent development. However, due to the diverse binding sites between antigens and antibodies, the high-scoring antibodies obtained through traditional methods, such as enzyme linked immunosorbent assay (ELISA), western blot, surface plasmon resonance (SPR) affinity detection, cannot fully represent their high sealing function towards the target protein. This uncertainty prolongs the research and development cycle and increases inspection costs. This article takes ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) as an example to introduce a patented (application number 202511470362.1, China) method for direct antibody screening at the functional level. This screening method mixes the ENTPD2 enzyme, substrate ATP and antibody in a strict dosage ratio to establish precise reaction conditions; upon completion of the reaction, the residual ATP content is detected to evaluate the inhibitory effect of the antibody on the enzyme. To ensure the accuracy of the screening process, we established cell systems and mouse models to verify the function of the antibodies. Using this method, we initially screened 5 antibodies from the supernatants of 214 rabbit monoclonal B cells. These 5 antibodies all demonstrated inhibitory function in both the cell system and the mouse model. These results show that this function screening method possesses the characteristics of high accuracy, high efficiency and high throughput, which can significantly shorten the research and development time and reduce cost.