A new method for rapid screening of hybridoma cell clones secreting paired antibodies using sandwich cell surface fluorescence immunosorbent assay.

ABSTRACT

Traditional methods of screening antibody pairs through ELISA-based methods are time-consuming and burdensome, which is not conducive for the rapid establishment of antigen detection methods. Hence, we developed a new method based on the sandwich cell surface fluorescence immunosorbent assay (SCSFIA) for rapid screening of paired antibodies. In this method, the capture antibodies were anchored to the hybridoma cells membrane through the lipid derivative Oleyl-PEG4000-NHS. Goat anti-mouse antibodies (blocking agent) were added to block the Fc fragment of the capture antibodies. The capture antibodies' Fab fragment can specifically bind the added antigen and form the capture antibodies-antigens complex (immunocomplexes). If the antibodies secreted by hybridoma cells could recognize the immunocomplexes. A double antibody sandwich structure would form on the cell surface based on the specific binding of antigens and antibodies. The hybridoma cells would be stained with anti-mouse IgG-Fc-FITC antibodies. We first used anti-pseudorabies virus (anti-PRV) cells and anti-porcine epidemic diarrhea virus (anti-PEDV) cells to verify the new method. Then, we used this method to successfully screen 5 hybridoma cell clones secreting paired antibodies against Avian influenza A (H7N9) virus within 15 days after fusion. These results showed that this method is suitable for the screening of paired antibodies in a variety of virus. Compared with the traditional method of obtaining paired antibodies, this method can greatly shortens the time needed to screen paired antibodies and improves screening efficiency, indicating that it is a promising method for paired antibodies discovery.