Cellular phenotype in primary screening

In collaboration with the American Red Cross, ACI has expanded a bead-based method of screening plasma-derived immunoglobulins for cytotoxic activity against melanoma cell lines. Using this method, thousands of antibodies are isolated and screened daily in an image-based assay that measures both cell number and propidium iodide (PI) fluorescence as an index of antibody-mediated phenotypic change.

Fig. 1. Five hundred WM266-4 human melanoma cells were plated to individual wells of a 384-well microculture plate. Beads conjugated with a random library of hexapeptides were incubated with patient plasma immunoglobulins and placed into the melanoma cell cultures at an average density of 30 beads per well. Culture medium included propidium iodide (PI) as an indicator of cell death. Visible and fluorescent red images of each entire well at ^40 magnification were taken after 72 h. The percentage ofPI-positive cells is plotted for each well. Two wells were treated with 50 pM etoposide as a positive control for cell death.

Fig. 1. Five hundred WM266-4 human melanoma cells were plated to individual wells of a 384-well microculture plate. Beads conjugated with a random library of hexapeptides were incubated with patient plasma immunoglobulins and placed into the melanoma cell cultures at an average density of 30 beads per well. Culture medium included propidium iodide (PI) as an indicator of cell death. Visible and fluorescent red images of each entire well at ^40 magnification were taken after 72 h. The percentage ofPI-positive cells is plotted for each well. Two wells were treated with 50 pM etoposide as a positive control for cell death.

Briefly, randomly generated libraries of six-member amino acid chains are synthesized on individual polystyrene beads, with each bead containing multiple copies of a single hexamer. These beads are incubated with human plasma to allow binding of plasma antibodies in an antibody-hexamer-specific manner. Plasma is obtained from patients in various stages of melanoma disease progression. In this reaction, antibodies are effectively purified by affinity interactions with the bead-linked hexapeptide. Beads are then rinsed and multiple beads are placed into 384-well microplate cultures of target cells. Bead-bound antibodies dissociate into the culture medium over time, forming an equilibrium between free and bound antibody.

Free antibody can bind to cellular targets, and cellular responses to antibody effects are detected by time-lapse digital imaging and quantified using image analysis. In this way, each well of the culture plate becomes a microbioassay system in which many antibodies are tested directly from plasma in a relatively purified form. Plates include control wells with antibody-free beads, and beads from wells showing statistically significant effects are removed from the well. At this point, one or more of this group of "hit well" beads is known to be loaded with an antibody already demonstrated to have a functional role in a disease-related bioassay.

Sample data from a single 384-well microculture plate assay are shown in Fig. 1. Melanoma cells were cultured with beads that had been incubated with melanoma patient plasma immunoglobulins for 3 d. Cells in more than 300 wells containing an average of 30 beads per well were imaged for visible and red fluorescent light. The total number of cells present as well as those that fluoresced red because of PI incorporation was determined using image analysis software. Wells showing increased levels of cell death when compared with wells containing unloaded, antibody-free beads are clearly identifiable.

Beads in positive primary assay wells are deconvoluted into separate wells, at a density of one bead per well, containing the same target cell line used in the multibead well. In this manner, the peptide-antibody combination responsible for the original functional response is both identified and confirmed in identical bioassays. The hexamer responsible for isolating the active antibody might or might not represent an actual linear portion of the B-cell epitope specific for the antibody. However, the peptide is a proven affinity ligand for the isolation ofthe antibody. Peptides from positive beads are sequenced by standard methods, and large-scale preparations of beads containing this peptide are synthesized. These scaled-up beads are, in turn, used to isolate large-scale preparations of antibody from plasma for secondary screening, in vivo studies, and monoclonal antibody sequencing to enable identification and cloning.

The value of an image-based cell death assay is apparent when considering that some drug candidates might cause cytostasis, only reducing cell proliferation without actually causing cells to die. Commonly used single-readout assays, such as annexin staining or caspase activation, fail to detect cytostasis if not coupled with a means of measuring absolute cell number. A simple comparison of cell numbers at the beginning and end of an assay period, such as that afforded through image analysis, is less likely to yield false negatives arising from candidates that act through annexin (1,2) or caspase-independent pathways (reviewed in refs. 3 and 4). The importance of simply observing cell cultures cannot be underestimated. With various cell/compound combinations, Automated Cell scientists have detected dividing cells that stained positive for either PI or annexin (unpublished observations and reviewed in ref. 5). The automated nature of the ACI platform allows for screening of millions of antibody-bead combinations at the phenotypic level on a weekly basis. Thus, the sensitivity of image-based cellular assays does not compromise the efficiency of the discovery engine for target discovery and validation.

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