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A Mix-and-Read Cell-Based Assay for Hybridoma Screening Using the FMAT ,,, 8100 HTS System

Abstract

The generation of hybrid myeloma cell lines for monoclonal antibody production is a fundamental and well-established technique. However, the tedious methods used to screen for specific antibodies secreted by hybridomas has varied little over the years. Screening for antibodies directed against cell-surface antigens is particularly problematic. We have developed a robust mix-and-read, cell-based assay for screening hybridoma supernatants using the fluorometric microvolume assay technology with the FMAT 8100 HTS System. The FMAT system employs a unique macroconfocal scanning platform to visualize and quantify both cell- and bead-based fluorescence in 96- and 384- well formats. Cells expressing the surface antigen were incubated with 5 L of hybridoma supernatant and were fluorescently-labeled with anti-mouse IgG antibodies. Without washing away unbound antibodies, plates were scanned and the positive wells were easily identified. A comparison with a multi-step cell ELISA done in parallel demonstrated that the fluorometric microvolume assay is the more sensitive and reliable method for hybridoma screening.

Introduction

Monoclonal antibodies are produced by hybrid myeloma or hybridoma cell lines that secrete specific antibodies into the growth media. The screening of hybridoma supernatants for specific antibodies is a critical component of monoclonal antibody generation. Conventional ELISAs, while tedious and time consuming, are sufficient when searching for antibodies directed against soluble antigens. However, screening for antibodies directed against cell-surface antigens is often difficult by cell ELISA. Cell loss during
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