Hybridoma screening using an amplified fluorescence microassay to quantify immunoglobulin concentration

The early identification of antibody-secreting hybridomas significantly reduces the time and resources devoted to unproductive colonies. To quantify the immunoglobulin concentration in hybridoma supernatants within 2 weeks of fusion, we used immunomagnetic microspheres to capture immunoglobulin in the hybridoma culture supernatant. The captured immunoglobulin was detected using a goat anti-mouse second antibody liked to beta-galactosidase. With data transformation to correct for the nonlinear accumulation of the fluorescent reaction product, the enzymatic hydrolysis of fluorescein digalactoside permitted the reliable detection of less than 10 pg of immunoglobulin per milliliter. To determine the value of quantifying immunoglobulin concentration within 2 weeks of fusion, the amplified fluorescence microassay was applied to the evaluation of 3 consecutive fusions and more than 1200 growing hybridoma colonies. Using antibody concentrations greater than 10 ng/ml as a threshold for routine subculture, the selection of hybridoma colonies based on antibody secretion was threefold more efficient than selection based on colony growth alone. These results suggest the utility of the early determination of immunoglobulin concentration in the selection of hybridoma colonies.