Revvity, 6FRARPAG, HTRF CD32a (131R) Cellular Binding Kit, 100 Assay Points

The CD32a (FcgRIIa, R131) cellular binding assay is a simple and robust test to monitor binding of the IgG Fc region to the human CD32a receptor in live cells and to select the most potent antibody for use in Antibody-Dependent Cell-Phagocytosis (ADCP). Two separate CD32a assays are available to specifically target the H131 or R131 variants. These assays, based on our innovative Tag-lite technology, can easily be automated to create a high-throughput solution for predicting ADCP. HTRF assays offer many advantages over other technologies: Homogeneous add-and-read format No wash steps Low background Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates Stable signal, providing flexibility in time of readout or size of assays
Assay principle The CD32a receptor cellular binding assay is a competitive assay involving HEK293 cells expressing a specific CD32a variant conjugated to Lumi4-terbium Cryptate, and human IgG1 labelled with d2. The unlabeled antibody or Fc tagged drug competes with the d2 labelled IgG1 for binding to the receptor, resulting in a decrease of FRET signal. Assay protocol The CD32a receptor cellular binding assays (FcgIIa H131 or R131) present only 1 incubation step after reagents distribution. Lumi4-terbium Cryptate pre-labelled cells are dispensed, followed by the addition of IgG1 labelled with d2 and the sample. After 2h incubation at RT, the signal can be read.