Efficient hybridoma screening using extracellular vesicles for conformation-specific antibodies to transmembrane proteins.

Here, we report a simple and rapid extracellular vesicle (EV)-based membrane-type immunoglobulin-directed hybridoma screening (MIHS) platform that enables efficient isolation of monoclonal antibodies (mAbs) recognizing three-dimensional conformations of transmembrane proteins. Transmembrane proteins are essential membrane components involved in signal transduction, transport, and energy conversion. As they function in native conformations, isolating mAbs that recognize conformational epitopes is critical for both research and therapeutic use. However, traditional methods, such as the enzyme-linked immunosorbent assay (ELISA), can disrupt the antigen structure, making it challenging to screen for such mAbs. To overcome this limitation, we developed a screening strategy combining MIHS with EVs as antigen-presenting tools. EVs, which are naturally bounded by lipid bilayers, display transmembrane proteins in native conformations and can be easily isolated. As a model, we engineered cells co-expressing alkaline phosphatase (ALP) and green fluorescent protein, generating fluorescent EVs displaying ALP. Hybridomas were screened by MIHS using these EVs, followed by ELISA without direct antigen immobilization. All isolated mAbs recognized conformational ALP epitopes, demonstrating the structural selectivity of this method. These results demonstrate that the EV-based MIHS approach provides a convenient and structurally faithful strategy for isolating conformation-specific mAbs, offering broad utility in basic biology and antibody development.