( Abstract The ...)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 the many wash steps of the procedure is a significant source of variability in a cell ELISA. An alternative to a cell ELISA is flow cytometry, which detects cell-surface staining by specific antibodies. However, flow cytometric analysis routinely involves handling and/or analyzing each sample individually in addition to numerous wash and incubation steps. Since thousands of clones are routinely screened, flow cytometry is not a practical option for high-throughput hybridoma screening. An ideal assay for hybridoma screening would be one that combines the reliability of flow cytometry with the plate format of a cell ELISA and can be performed without wash steps. The FMAT 8100 HTS System provides a unique platform that is perfectly suited for hybridoma screening. The FMAT system is a macroconfocal scanner that visualizes and quantitates both cell- and bead-bound fluorescence. Light generated from a 633 nm helium/neon laser scans an area of 1 mm2 with a depth of focus of ~100 m from the bottom of each well. Because the depth of focus is relatively small with respect to the remaining volume of the well, the majority of the unbound fluorophores remains undetected and need not be washed away. The FMAT software is then able to subtract the remaining background fluroscence from the fluorescence immobilized on the cells or beads. As a result, all assays performed with the FMAT system are mix-and-read and do not require wash steps. The integrated plate handler allows for walk-away screening for up to sixty 96- or 384- well plates. A screen for a monoclonal antibody directed against a cell-surface antigen was developed using the FMAT system. Each well contained cells expressing the cell-surface antigen of interest, FMAT Blue dye-labeled anti-mouse IgG, and as little as 5 L of hybridoma supernatant. A total of 51 positive wells from 14 plates were easily and unambiguously identified by the FMAT system using a simple mixand- read assay. A cell ELISA screen performed simultaneously with the fluorometric microvolume assay generated inconsistent data that was inconclusive. A select number of hybridoma supernatants that were identified as positive by the FMAT system were confirmed by flow cytometry, establishing that the single- step fluorometric microvolume assay is a simple yet robust method for cell-based hybridoma screening.

Methods

FMAT 8100 HTS System Assay Cells expressing the cell surface antigen were grown in tissue culture flasks, collected, washed with screening buffer (PBS, 2% FCS), and resuspended in screening buffer at a concentration of 1x105 cells/mL. FMAT Blue dye-labeled goat antimouse IgG(Fc) was diluted to 0.4 g/mL in screening buffer. To initiate the hybridoma screen, 100 L of cells, 50 L of the labeled anti-mouse antibody and 5 L of hybridoma supernatant were added to each well of 14 FMAT system 96-well plates. No mixing was required. The final volume of 155 L per well contained 10,000 cells and 0.13 g/mL of detection antibody. The plates were scanned after 2 hours of incubation. A well was considered positive if it had a count of over 50 events. A simultaneous screen using control cells (without antigen expression) was performed as described in Figure 1.

Cell ELISA Cells expressing the cell surface antigen were grown in tissue culture flasks, collected, and plated in 14 96-well plates. After incubating for 48 hours, the tissue culture supernatant was removed and the wells were blocked with 200 L of screening buffer for 1 hour. The buffer was removed and replaced with 5 L of hybridoma supernatant and 95 L of fresh buffer. After 1 hour of incubation, the supernatant was removed and the wells were gently washed 6 times with buffer. Detection antibody (goat anti-mouse IgG (Fc)-HRP) was added, followed by an additional hour of incubation. The supernatants were removed, the wells were gently washed 6 times with buffer and substrate was added. The color was allowed to develop for 5 to 10 minutes before stopping reagent was added. Plates were read using a standard plate reader.

Results

Out of a total of 1,344 wells, 51 were considered positive by the FMAT 8100 HTS System. The positive wells were further subdivided into those with low (< 500: 32 wells), medium (500-1,000: 14 wells), or high (>1,000: 5 wells) average fluorescence intensity (FL1) values.

A simultaneous screen using control cells that do not express cell surface antigen did not yield any hits. The FMAT software allows the results to be viewed in several different formats (Figure 1, panels A-D) for simple and unambiguous selection of positive wells. In addition, a tab-delineated file containing a summary of the data from all fourteen plates and in 96-well grid format can be accessed. A detailed well view allows for real-time or postscan evaluation of the positive wells (Figure 2). A cell ELISA screen was performed in parallel in an attempt to confirm the data obtained with the FMAT system. However, the data from the cell ELISA was inconclusive, prohibiting any meaningful comparisons. For example, the FMAT system easily identified 6 positive wells from one plate of the screen (Figure 3, panel A). However, only one positive well is evident from the same plate assayed with a cell ELISA (Figure 3, panel B). The remaining wells identified as positives by the FMAT system are obscured by the high fluorescent background in the cell ELISA plate. Another factor influencing the outcome of the cell ELISA was the high degree of variability in the number of cells per well, resulting in unreliable comparative data. Although great care was taken, cells were nonetheless lost during the many wash steps because of the cells poor adhesion properties. Because no wash steps are required for the fluorometric microvolume assay, variability in the data due to cell loss is not a concern. Since a direct comparison between the FMAT system and the cell ELISA was not feasible, several of the positive wells identified by the FMAT system were analyzed by flow cytometry (data not shown). In all cases, the FMAT system and the flow cytometry data were in agreement with respect to identification and quality of the hits.

An additional advantage of the FMAT system over the cell ELISA is the ability to visually inspect the wells. Images of the positive wells from the same plate represented in Figure 2 are depicted (Figure 3). The fluorescence intensity observed on the cells matched the average FL1 values obtained for those wells. On rare occasions, a false positive may occur if a well contains an abundance of fluorescent debris. By inspecting the images an observer can easily see the debris and remove that well from consideration. Levels of IgG were determined for several of the positive wells and one negative well.

Although some of the wells with high average fluorescence intensity values contain high levels of IgG (e.g. wells 2E11, 6A10), this correlation does not extend to all of the wells (e.g. wells 2A7, 6B5). The quality of the antibody-antigen binding reaction can therefore be evaluated independently of the amount of murine IgG secreted into the media. Indeed, reliable data can be obtained for wells containing as high as 22 g/mL IgG to as low as 0.16 g/mL IgG.

Conclusion

The hybridoma screening assay developed for the FMAT system was simple to develop, set-up, and execute. Cells for the fluorometric microvolume assay were plated just minutes prior to the screen, unlike the cell ELISA where cells had to be plated 48 hours prior to the screen. This eliminates any edge effects due to uneven cell growth across the plate and reduces cell culture burden. Because of the mix-and-read format of the assay, all cell types (suspension or adherent) can be used without further manipulations. With a cell ELISA, suspension or weakly adherent cells must be fixed by some means to the well surface and thereby introduces potentially detrimental variables to the screen. Besides cell-surface antigens, the FMAT system can be used to screen for monoclonal antibodies directed against soluble antigens by using polystyrene beads coated against the antigen (Figure 5). With both the cell- and bead-based assays, no wash steps and only one incubation step are required. Moreover, only 5 L of the hybridoma supernatant is required for the assay, enabling parallel multi-specificity screening at the fusion plate stage. In contrast, the cell ELISA typically requires 50 to 100 L of hybridoma supernatant. In addition, the FMAT system allows for multiplexed assays based on bead size or dye color, enabling more than one assay to be performed in a single well. With its ease-of-use, flexibility and sensitivity, the FMAT system is ideally suited for hybridoma screening.
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