Dual-color membrane-type immunoglobulin-directed hybridoma screening for selective and accelerated monoclonal antibody production.

Monoclonal antibodies (mAbs) are essential tools in life sciences and medicine because they provide high specificity and selective binding properties. However, their production is labor-intensive. Conventional hybridoma screening using the limiting dilution method requires repeated manual handling and long-term culture, which slows the isolation of target antibodies. To facilitate this process, we previously developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method that directly detects interactions between membrane-bound B-cell receptors and fluorescently labeled antigens. However, the preparation of multiple soluble antigens remains a challenge. Hence, we designed a single recombinant antigen, X-His-BAP-MBP (X-HBM), in which the target antigen (X) was fused to a solubilization tag (MBP), biotinylation tag (BAP), and His-tag for purification. We established a dual-color MIHS system that integrates immunization, screening, and evaluation into a workflow. Dual-color fluorescence labeling enabled the discrimination and exclusion of hybridomas producing antibodies against MBP or tag sequences, allowing the precise isolation of hybridomas secreting antibodies specific to the target antigen. Using the mitochondrial enzyme CARS2 as a model antigen, the proportion of target-positive hybridomas increased approximately eightfold compared to that using the conventional method. Specific mAbs were successfully obtained using only a single soluble antigen preparation through the MIHS method. This method supports the efficient selection of antigen-specific hybridomas, even when solubilization tags are used, by achieving both antigen specificity and practical solubility.