Assay format

Cell-based assays

Cisbio Bioassays presents a wide selection of cell-based assays developed with HTRF® technology. HTRF® assays listed in this section were developed to facilitate working with whole cells, thereby enabling functional assays to be run under physiological conditions. For HTS purposes, HTRF® cell-based assay protocols have been streamlined to single-plate processes from cell culture to assay readout. Cells remain alive until the final detection step.

Features

• Truly homogeneous functional assays
• Mix-and-read protocol
• Flexible assay procedures:
  -Assessment of secreted molecules via their quantification in cell culture medium, even when complemented with FCS
  -Simultaneous cell lysis and detection to assess intracellular messengers using a single-plate protocol
• Time-resolved detection to correct for prompt background fluorescence
• Ratiometric correction to avoid medium interference

Example of an HTRF® cell-based assay:

Sandwich assays

These assays are designed to assess molecules that are large enough to encompass two distinct binding sites for two antibodies (Ab). As shown here, HTRF® sandwich assays use two antibodies coupled respectively to Eu3+ or Lumi4®-Tb cryptate and to an HTRF® acceptor (i.e. XL665 or d2). The two conjugates bind to the antigen when present in the sample, thereby generating FRET.

Immunocompetitive assays

These assays are designed to assess molecules that are too small to allow the binding of two antibodies. The two assay components used here consist of an acceptor conjugate generally made of purified antigen, and its respective Ab coupled to Eu3+ or Lumi4®-Tb cryptate. The Ag present in the sample competes with the binding between the two conjugates and thereby prevents FRET from occurring.