Assay format

HTRF®, a technological platform for your own assay development

HTRF was originally created as an immunoassay technology nearly two decades ago, and continues to offer one of the most effective and flexible solutions in the diagnostic field today. Its versatility has led to the optimization of numerous assay configurations for drug research, from the most basic biochemical tests to more sophisticated cell-based assays. HTRF has become a reference technology for researchers who wish to transition from conventional assays such as Western Blot, ELISA, or bead-based technologies, and successfully convert to fast and cost-effective mix & measure assay formats.

 

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.

The following list shows a selection of assays based on an immunometric format (sandwich assays)

Assay
Cell-based phospho-protein
(pErk, pAkt, etc.)
Cytokines
(h&m TNFa, IL2, IL8, etc.)
Apo A1 and B
Insulin
Amyloid B1-40
Human IgG Kappa and Lambda

Signal intensity is proportional to the number of Ab-Ag complexes formed, and therefore to Ag concentration. The specific signal modulates positively in proportion to Ag concentration.

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.

The following list shows a selection of assays based on an immunocompetitive format

Assays
Aldosterone Histamine
cAMP IP-One
CD16a LTB4
cGMP LTC4
Cortisol PGE2
Estradiol SIRT1
Glucagon Total human igG
GST and 6HIS check kits  

FRET intensity decreases with the amount of dissociated Ab-Ag complexes. The specific signal modulates nega- tively, is therefore inversely proportional to Ag concentration.