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

• Screening for Ab-secreting hapten-specific hybridoma cells
• Double staining of surface IgG by fluorophore labeled haptens and antibodies
• Double staining verified by confocal laser scanning microscopy
• Flow cytometry intensities consistent with IgG amount, selectivity and affinity
• ELISA sensitivity and selectivity of secreted IgG correspond with flow cytometry.

A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.

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