Revvity, 64C1QPET, HTRF Human C1q Binding Kit, 100 Assay Points

Overview
Complement-Dependent Cytotoxicity (CDC) is an important effector function of antibodies. Activation of the classical complement pathway is initiated by the binding of the complement protein C1q to the Fc region of immunoglobulins bound to a cell surface target antigen. This interaction triggers the sequential enzymatic activation of circulating complement proteins, and finally leads to the formation of the Membrane Attack Complex (MAC) that mediates target cell lysis.
The capacity of C1q binding and resultant CDC activity can influence the safety and efficacy of therapeutic monoclonal antibodies, and therefore requires characterization during the development process. Engineering the Fc region of IgG antibodies (e.g. by mutations or glycoengineering) is currently an active area of investigation to enhance or reduce the ability of biologics to recruit complement.
The human C1q binding kit measures the binding of the Fc region of aggregated IgG antibodies to human C1q in solution.
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
How it works
HTRF Human C1q Binding assay principle
The HTRF Human C1q binding assay measures the binding of the Fc region of IgG antibodies to human C1q in buffered solution. Unlike conventional methods that require the immobilization of antibodies on a solid phase, this assay enables simple and rapid characterization of antibodies in a homogeneous generic format. A biotinylated anti-human IgG Fab antibody complexed to streptavidin is used to capture and aggregate the tested antibody in solution. Antibodies bound to human C1q are detected in a sandwich assay format using an anti-C1q antibody labelled with Eu3+ cryptate (donor), and the streptavidin labelled with d2 (acceptor). The interaction between the two partners brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the capacity of the antibody Fc region to interact with human C1q.

 

HTRF Human C1q Binding assay protocol
The human C1q binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). It requires only 1 incubation step after the sequential addition of assay reagents. As described here, tested antibodies (or the standard) are dispensed directly into the assay plate. The premix [Anti-Human IgG-Fab-biotin + Streptavidin-d2] is then added, followed by the dispensing of the human C1q protein and the anti-C1q antibody labelled with Eu3+ Cryptate. After a 3h-incubation at RT, the FRET signal can be recorded on an HTRF compatible reader. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally. Application-Protein-Protein Interaction
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Sample Volume-5 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Binding Assay
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology & Inflammation
Unit Size-100 assay points