The use of cultured cells for the assessment of compound activity offers many advantages over in vitro biochemical assays. Cellular models allow the simultaneous probing of various signaling pathways and the evaluation of a drug’s permeability to cellular membranes. An End-Point Assay using Enzyme-Linked Immunosorbent Assays (ELISA) is the most widely adopted method for biomarker detection and quantification. ELISA offers high selectivity, sensitivity and assay versatility; however, it presents certain limitations such as a narrow dynamic range, low throughput and modest reproducibility due to its numerous wash steps.
In contrast, homogeneous chemiluminescent bead-based AlphaLISA® assays allow measurement of biomarkers in a high throughput mode in the absence of any wash step. In the present work, various cytokines (TNFα, IL1ß, IL6, and IL8) and an integral plasma membrane protein (EGFR) were measured on stimulated cells using an All-In-One-Well AlphaLISA assay format. Biomarker production was measured from the adherent cell lines A431 or A549, and from suspension THP-1 cells directly in 384-well culture plates, in the absence of any transfer or wash step. This technology simplifies cellular assays, significantly reduces hands-on time and costs associated with plastic ware, and improves reproducibility. Moreover, results show excellent assay performance with wide dynamic range, low interference from cells or cell culture media, and high sensitivity. Indeed, AlphaLISA technology is a userfriendly and versatile tool for generating immunoassays for cellular models.
TNFα measurement. A) For the standard curves, TNFα was spiked at different concentrations in wells containing only medium or non stimulated THP-1 cells, and B) cells in log phase were plated and stimulated with different concentrations of LPS. All AlphaLISA reagents were added to the same well for the detection and TNFα concentrations were determined from the standard curve.