Section8:Introduction

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Cell-based ELISA/Western Flow Chart: Overview and Practical Considerations

Concept

The cell-based ELISA or Western (cell-based ELISA or C-ELISA) is a moderate throughput format for detecting and quantitfying cellular proteins including post-translational modifications associated with cell activation (e.g., phosphorylation and degradation). Typically, these changes are monitored by Western blots. However, this procedure requires cell lysis, electrophoresis, blotting and staining of the gel with the appropriate antibody. Western blots are only semi-quantitative and have very low throughput. The C-ELISA does not require cell lysis, electrophoresis of the sample or membrane blotting. The C-ELISA does allow detection and quantitation of specific cellular proteins in a 96 well plate format. Furthermore, the C-ELISA is amenable to automation that facilitates the screening of large numbers of compounds.

In the past several years there has seen an explosion in the availability of commercial sources of antibodies to cell signaling molecules. In addition, antibodies that selectively recognize post-translationally modified proteins (e.g., phosphorylated, acetylated) have also become available. In many cases, these antibodies are very high quality as determined by strength of signal and specificity on immunoblots. These same high affinity, specific antibodies can also be used to detect antigens in fixed cells by immunofluorescence. The spatial and temporal information derived from these studies can be extremely valuable in delineating the function of post-translational modifications. For example, phosphorylation can be a trigger for a change in subcellular localization and consequently, a change in protein function.

Unfortunately, for the purposes of high throughput drug discovery, both immunoblots and immunocytofluorescence have severe restrictions. Immunoblots are not readily adaptable to 96-well plate formatted experimental designs. Although chemilumescent detection systems have made immunoblots extremely sensitive, quantitation is limited by the small linear range inherent in exposed film. More quantitative methods using newer instrumentation (e.g., phosphoroimagers) alleviate some of these difficulties; however, the laborious procedures for preparing cell lysates, determining protein concentrations, loading gels and blotting remain. Immunocytofluorescence with conventional microscopy is also tedious, relatively insensitive and non-quantitative. In order to take advantage of the high quality antibodies available for studying cell-signaling pathways, we have developed procedures for combining the best properties of immunocytofluorescence and immunoblots. This cell-based western (also referred to as cell-based ELISA or C-ELISA) has proved to be extremely useful for medium throughput screens of kinase inhibitors and, in conjunction with biochemical assay data, has become an important SAR driving force.

The key ingredient for a successful cell-based Western assay is a high quality antibody. Single band antibody specificity must first be established by conventional immunoblot procedures. Once a suitable antibody is identified, it can be used to stain and quantitate the levels of antigen in cells in individual wells of a 96-well plate. Cells are plated, treated according to experimental requirements and fixed directly in the wells, similar to an immunocytofluorescence experiment. After fixing, individual wells go through the same series of steps used for a conventional immunoblot, including blocking, incubation with first antibody, washing, incubation with second antibody, addition of chemilumescent substrates and development. Finally, results are read in a luminescence plate reader. Other readouts including fluorescence can be used in this assay format. In our experience, this procedure provides rapid, quantitative analysis of cellular antigens. In general, the correlation between immunoblot and C-ELISA assays is quite good (Appendix 1). The wide linear range of the C-ELISA allows for quantifying >5-fold changes in cellular protein levels in response to stimulant (Appendix 2). The versatility is only as limited as the availability of high quality antibodies to the target proteins.

Cell-based ELISA/Western Schematic