As a part of the innate immune system, the complement system serves as an important auxiliary of the immune system in enhancing microbe clearance and inflammatory reactions. Generally, the complement system is activated and functions through three biochemical pathways: the classical complement pathway, the alternative complement pathway, and the lectin pathway, in which the classical pathway of the complement system is considered to be the most important pathway in immune complex clearance.
The classical complement pathway is usually initiated by antigen-antibody complexes through the binding of C1q to IgM or IgG complexed with antigens. As a subcomponent of the first component (C1) of the classical pathway, C1q mainly binds to the Fc domain of IgM or IgG antibodies (the CH4 domain of IgM and the CH2 domain of IgG except for IgG4). Once binding of C1q to the antibody of the immune complex, the C1 complex becomes activated, so do the classical complement pathway and complement-dependent cytotoxicity (CDC).
Fig.1 Activation of the classical pathway of complement. (Frachet, 2015)
The engineered therapeutic monoclonal antibodies (mAbs) with Fc fragment modification to enhance or reduce the complement activities, such as mutations or glycosylation engineering, have raised and accounted for an increasing proportion. The binding ability of developed antibodies to complement C1q and resultant complement classical cascade activation is an important aspect for measuring the efficacy and safety of therapeutic mAbs. And therefore, complement C1q-binding assay is necessary for characterization during the development process of therapeutic mAbs.
Complement C1q-binding assay is mainly used for measuring the ability of antibody candidates binding to the complement C1q. For the assay, antibody samples developed by clients with different concentration gradients will be tested, and proper positive and negative controls will be included. Please mark the concentration and volume of the antibody samples when you ship the samples to us.
Semi-quantitative enzyme-linked immunosorbent assay (ELISA)
1-2 weeks. The test time may vary depending on the number of antibodies, and additional time should be allowed for additional validation testing or repetition, if necessary.
intuitive. During the tests, the binding of test articles to the secondary antibody will be verified first. Then, detection of the binding of samples with different concentration gradients to complement C1q by ELISA titration. EC50 of each sample will be presented and proper positive and negative controls will be included.
Fig.2 Workflow of Complement C1q-Binding Assays. (Creative Biolabs)
Fig.3 Complement C1q binding assay performed by ELISA titration. (Creative Biolabs)
The clients sent us 3 engineering antibody samples to characterize the binding ability of their antibody samples to complement C1q. We first perform the validation of the binding of samples to the secondary antibody, and found that all the samples had normal binding with the secondary antibody. Then we perform the complement C1q binding assay of samples by ELISA titration through setting 9 different concentration gradients. Binding curves and EC50 (not shown) of each sample were obtained based on the ELISA OD450 value. And the results indicated that all 3 samples had a normal binding to complement C1q.
As a leading service biotech company, Creative Biolabs has armed with a powerful complement test platform, aiming to offer the best and most convenient complement-related services. Over the last 20 years, Creative Biolabs has completed many customers' projects, which have allowed us to develop proprietary custom services that are unrivaled in the industry. We will be your reliable and trustable partner featured with:
Creative Biolabs undertakes C1q-binding assays on behalf of clients as standalone projects or as part of larger development projects. Our focus is to exceed your expectations while providing accurate and reliable complement test services. To get more information, a quote, or to schedule a teleconference, please contact us.
Reference
A: More than 50 methods have been described for the assessment of immune complexes in serum or plasma. Measurement of circulating immune complexes (CIC) in the serum of patients with different diseases is often used to assess disease activity for disease management, treatment and control purposes. Complement C1q binding assays are the most widely used to measure CIC. Using the property of ICI to bind to C1q, it is possible to detect immune complexes that activate complement in the classical pathway.
A: C1q is part of the C1 complex, which is the first complement component of a cascade reaction called the classical complement pathway. C1q has six extended arms, each with a structural domain at the end that binds to the Fc structural domain of an immunoglobulin. When antibodies bind to antigens to form an immune complex, they come together so that two or more of the six arms of C1q bind to the Fc structural domain of an antibody, such as IgG or IgM.
A: (a) Liquid phase method. Using isotopically labeled C1q mixed with inactivated serum specimens. (b) Solid-phase method. C1q is adsorbed on the surface of the solid phase carrier, and the serum to be tested and isotopically labeled or enzyme-labeled anti-human IgG are added. (3) Hemolysis test. Mix isotopically labeled C1q with inactivated serum specimens and add antibody-sensitized sheep red blood cells.
A: The sample volume requirement may vary depending on the sensitivity of the assay and the concentration of your sample, but generally, we require a minimum sample volume of 1 mL for the C1q-binding assays. Detailed information about your samples such as sources, concentration, and preparation methods is very helpful to ensure the accuracy of the assays. You should also inform us of any special handling considerations.
A: The results report will provide a comprehensive overview of the study. This includes detailed analysis of your samples' C1q-binding capabilities, raw data and interpretation, plus any observations or additional findings during the assay. We also offer customization for our assays to meet specific experimental requirements.
A: Our C1q-binding assays, in many instances, are designed to be very adaptable and can be used in high-throughput screening processes. This allows you to study several samples simultaneously, saving time and resources.