Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igα and Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igα expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).

Peripheral nerve injury results in a series of events culminating in degradation of the axonal cytoskeleton (Wallerian degeneration). In the time period between axotomy and cytoskeletal degradation (24-48 h in rodents), there is calcium entry and activation of calpains within the axon. The precise timing of these events during this period is unknown. In the present study, antibodies were generated to three distinct peptide epitopes of m-calpain, and a fusion protein antibody was generated to the intrinsic calpain inhibitor calpastatin. These antibodies were used to measure changes in these proteins in mouse sciatic nerves during Wallerian degeneration. In sciatic nerve homogenates and cultured dorsal root ganglion (DRG) neurites, m-calpain protein was significantly reduced in transected nerves very early after nerve injury, long before axonal degeneration occurred. Levels of m-calpain protein remained low as compared to control nerves for the remainder of the 72-h time course. No changes in calpastatin protein were evident. Systemic treatment of animals with the protease inhibitor leupeptin partially prevented the rapid loss of calpain protein. Removal of calcium in DRG cultures had the same effect. These data indicate that m-calpain protein is lost very early after axonal injury, and likely reflect activation and degradation of this protein long before the cytoskeleton is degraded. Calpain activation may be an early event in a proteolytic cascade that is initiated by axonal injury and culminates with axonal degeneration.