Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas

Abstract

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 Igαand Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating an order of magnitude increase 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).

Anti-Human Embryonic Stem Cell Monoclonal Antibody Hesca-2 Binds to a Glycan Epitope Commonly Found on Carcinomas

“…Antibody secreting hybridoma cell lines were isolated by fluorescent-activated cell sorting (FACS) based on expression of the membrane isovariant of that antibody in the B cell antigen receptor (BCR) complex on the cell surface using a proprietary process described previously [16]. Briefly, this process relies on fusing splenocytes with a myeloma (SP2ab) that has been genetically engineered to constitutively express the Iga and Igb (CD79a and CD79b) components of the BCR.…”

“…Hybridomas were removed from culture, washed, labeled with biotin-conjugated PMA fractions, and counterstained with streptavidin-conjugated r-phycoerythrin (BD Biosciences) and allophycocyaninconjugated goat anti-mouse Ig (Invitrogen). Sorting was done using a FACS AriaÔ Flow Cytometer (BD Biosciences) as described previously [16]. Cells positive for both Ig expression and PMA reactivity were deposited singly into 96-well culture plates containing Iscove's modified Dulbecco's media supplemented with 20% fetal bovine serum, hypoxanthine, and thymidine (Sigma-Aldrich).…”

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

“…From these findings we conclude that our optimized immunofluorescence staining procedure is specific. Price et al, 2009 showed different amounts of cell surface IgG on transfected cells by immunofluorescence microscopy. However, microscopic images of antigen-specific antibodies on the surface of hybridoma cells have not been shown in the literature so far.…”

“…In another context genetically modified hybridoma cells were sorted antigen-specifically via a streptavidin/biotin system (Price et al, 2009).…”

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody

“…2A). SP2/0 murine myeloma cells that express endogenous Igb 16 were transfected with vectors containing sequences encoding the individual constructs, and surface expression of SM03 binding moiety was examined by flow cytometry (Fig. 2B).…”