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HTRF Human C1q Binding Kit, 100 Assay Points

The Human C1q binding kit is designed to monitor the binding of the IgG Fc region to the human C1q protein.

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Feature	Specification
Application	タンパク質間相互作用
Sample Volume	5 µL

The Human C1q binding kit is designed to monitor the binding of the IgG Fc region to the human C1q protein.

View product information

Product variants

Unit Size: 100 assay points

Part #:

64C1QPET

Unit Size: 500 assay points

Part #:

64C1QPEG

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

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.

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.

Principle of the HTRF human C1q binding assay

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.

Assay validation

Binding profiles of human IgG isotype controls

Purified human IgG1, IgG2 and IgG4 isotype control antibodies were serially diluted with Diluent #12 and tested in the Human C1q binding assay. The signal intensity obtained for each dose-response binding curve shows that human IgG1 binds efficiently to C1q, and human IgG2 interacts weakly with the complement component, whereas human IgG4 does not bind to the protein. These results are in accordance with the literature (Almagro et al., 2018, Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy. Front. Immunol. 8:1751).

Validation of human C1q binding assay with human IgG isotype controls

Binding profiles of Rituximab variants

The therapeutic anti-CD20 antibody Rituximab (chimeric IgG1) was tested in the Human C1q binding assay in parallel with the following Rituximab variants: non-fucosylated Rituximab (InvivoGen, #hcd20-mab13), non-glycosylated Rituximab (InvivoGen, #hcd20-mab12), Rituximab IgG2 isotype (InvivoGen, #hcd20-mab2), and Rituximab IgG4 isotype (InvivoGen, #hcd20-mab4). As expected, the absence of the fucose residue on the Fc domain of the non-fucosylated antibody (supposed to modulate ADCC activity only) did not induce any detectable change in C1q binding compared to Rituximab. Conversely, the capacity of interaction of the non-glycosylated variant was altered, demonstrating that antibody Fc glycosylation is essential for Fc-mediated effector mechanisms. Finally, isotype switching from IgG1 to IgG2 or IgG4 induced a considerable decrease or an elimination of the capacity of the antibody to bind to C1q.

Validation of human C1q binding assay with Rituximab variants

Binding profiles of therapeutic type I & type II anti-CD20 antibodies

The therapeutic anti-CD20 antibodies Rituximab (type I, chimeric IgG1) and Obinutuzumab (type II, humanized IgG1, also known as 'GA101') were tested in the Human C1q binding assay. The lower signal obtained with the type II anti-CD20 Obinutuzumab demonstrates its reduced capacity to bind to C1q compared to the type I anti-CD20 Rituximab. These results are consistent with literature (Herter et al., 2013, Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models, Mol Cancer Ther; 12(10); 2031–42).

Validation of human C1q binding assay with therapeutic type I & type II anti-CD20 antibodies

Binding profiles of Cetuximab variants

The therapeutic anti-EGFR antibody Cetuximab (chimeric IgG1) was tested in the Human C1q binding assay in parallel with the following Cetuximab variants: non-glycosylated Cetuximab (InvivoGen, #hegfr-mab12) and Cetuximab IgG2 isotype (InvivoGen, #hegfr-mab2). As expected, the binding capacity of the non-glycosylated variant was altered, demonstrating that antibody Fc glycosylation is essential for Fc-mediated effector mechanisms. Isotype switching from IgG1 to IgG2 also induces a considerable decrease in the capacity of the antibody to interact with C1q.

Validation of human C1q binding assay with Cetuximab variants

Binding profiles of Ipilimumab and IgG2 isotype variant

The therapeutic anti-CTLA-4 antibody Ipilimumab (fully human IgG1) was tested in the Human C1q binding assay in parallel with its IgG2 isotype variant (InvivoGen, #hctla4-mab2). The signal decrease observed between the biologic and its variant demonstrates that the isotype switching from IgG1 to IgG2 leads to a considerable loss of capacity of the antibody to bind to C1q.

Validation of human C1q binding assay with Ipilimumab and IgG2 isotype variant

Binding profiles of therapeutic anti-PD-L1 & anti-PD-1 antibodies

The therapeutic antibodies Atezolizumab (anti-PD-L1, humanized IgG1) and Spartalizumab (anti-PD-1, humanized IgG4) were tested in the Human C1q binding assay. As expected, the anti-PD-L1 IgG1 antibody Atezolizumab showed strong binding to C1q, whereas the anti-PD-1 IgG4 antibody Spartalizumab did not have the capacity to interact with C1q.

Validation of human C1q binding assay with therapeutic anti-PD-L1 & anti-PD-1 antibodies

Binding profiles of therapeutic anti-TNF-α antibodies

The therapeutic anti-TNF-α antibodies Infliximab (chimeric IgG1) and Adalimumab (fully human IgG1) were tested in the Human C1q binding assay. The binding dose-response curves showed similar profiles and signal intensity, demonstrating that both anti-TNF-α antibodies have the same capacity to interact with C1q.

Validation of human C1q binding assay with therapeutic anti-TNF-α antibodies

Specifications

Other specifications

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