Digestion of IgG Below the Hinge Region
CD BioGlyco provides systematic and one-stop Antibody Digestion Services for the digestion of IgG below the hinge region and ensures the continuity and transparency of the experimental process.
The Structure of F(ab')2 and Fc Fragments
It is very useful to study or exploit the partial activity of immunoglobulins without interference from other molecular fragments. Immunoglobulin molecules are selectively cleaved into fragments with discrete features.
F(ab')2 fragments refer to two antigen-binding F(ab) moieties linked together by disulfide bonds. Compared to whole IgG molecules, F(ab')2 fragments are smaller, allowing better penetration into tissues and facilitating better antigen recognition in IHC. The use of F(ab')2 fragments also avoids non-specific binding to specific receptors (Fc receptors) or specific proteins (Protein A/G) on living cells.
Fig.1 The structure of F(ab')2 fragments. (Bates & Power, 2019)
The fragment crystalline region (Fc region) mainly binds to Fc receptors on the cell surface and interacts with some proteins of the complement system. This property is closely related to the activation of the immune system by antibodies. Therefore, glycosylation of Fc fragments is critical for Fc receptor-mediated activity. In the IgG antibody isotype, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody.
Key Technologies
Our core technology is the use of IdeS (Immunoglobulin G-degrading enzyme from Streptococcus pyogenes). IdeS is a cysteine protease that specifically cleaves all subclasses of human IgG below the hinge region, generating F(ab')2 fragments and intact Fc fragments. We also utilize a suite of other proteases and custom protocols to accommodate various IgG subclasses, species, and unique project requirements, ensuring that every digestion is optimized for maximum yield and fragment integrity.
Digestion of IgG Below the Hinge Region at CD BioGlyco
CD BioGlyco provides an enzyme that digests IgG at a specific site below the hinge region under physiological reaction conditions, with optimal activity at 37°C. Digestion produces a homogeneous library of F(ab')2 and Fc/2 fragments. As far as we know IgG is the only substrate. In general, IgG is digested within half an hour, but not over-digested with longer incubation times.
Our high specific enzyme can digest all subclasses of human, monkey, rabbit, and sheep IgG, but it has limited activity on mouse IgG2a and IgG3. We will introduce related services on another page: Digestion of Mouse IgG2a and IgG3 Below the Hinge Region.
- Project Design
Clarify the core objectives of your project first: the specific IgG subtype targeted for digestion, the type of fragment you aim to obtain, and the intended downstream use scenarios. On this basis, determine the most suitable digestion approach, and screen out the optimal protease type and matching reaction parameters (such as temperature, pH, and reaction time).
- IgG Preparation
You may provide purified IgG samples directly, or we can support you in completing the IgG purification process. Subsequently, we will conduct a strict quality inspection to confirm two critical indicators of the initial material: the structural integrity and concentration.
- Enzymatic Digestion
We employ specific enzymes (e.g., IdeS protease) to carry out cleavage reactions. Throughout the process, reaction conditions are strictly monitored and controlled.
- Fragment Purification and Isolation
After the digestion reaction, we adopt efficient separation methods (e.g., Protein A chromatography) to isolate the Fc fragment. The core goal of this step is to completely remove impurities, including F(ab')2 fragments generated during digestion, unreacted intact IgG, and residual protease, thereby obtaining high-purity Fc fragments.
- Characterization
To fully confirm the quality of the prepared Fc fragment, we combine multiple analytical technologies for comprehensive detection: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is used to verify molecular weight and purity; mass spectrometry (MS) helps confirm structural identity; in addition, other professional bioanalytical methods are applied to further ensure the overall integrity and usability of the Fc fragment.
Workflow
Publication Data
Journal: Antibodies
IF: 2.7
Published: 2019
Results: The review found that smaller antibody fragments have several advantages over full-length mAbs, including lower production costs, higher yields, and better tissue penetration due to their small size. These properties allow them to reach difficult-to-access epitopes and are particularly useful for applications like cancer immunotherapy. These new formats offer exciting opportunities to expand the use of antibodies into previously challenging areas, such as developing oral antibodies for neurological diseases or targeting intracellular molecules.
Applications
- Modifying Fc glycans fine-tunes effector functions, leading to the development of new therapeutics with enhanced potency and safety.
- Using defined Fc fragments significantly improves the specificity and sensitivity of immunoassays, enabling the development of more reliable diagnostic tools.
- High-quality, homogeneous Fc fragments are essential for structural studies, helping to elucidate the role of glycosylation in antibody structure and function.
- Our fragments provide a unified platform for detailed glycan analysis, helping researchers understand the complex relationship between glycan structure and biological activity.
Advantages of Us
- One-stop service for digestion of IgG below the hinge region
- Customize experimental programs according to different research needs
- The high specificity of the enzymes used
- Responses under physiological conditions allow the preservation of the immunoreactivity of IgG
- Detailed and reliable after-sales service
Frequently Asked Questions
CD BioGlyco has an advanced Glycoengineering Platform in glycoscience. Our professional research teams allow us to provide you with high-quality services for the digestion of IgG below the hinge region. If you are interested in our services, please contact us for more details without any hesitation.
Associated Services
Generate specific glycan-modified proteins or conjugates for your research needs.
Engineer and produce antibodies with specific Fc regions, including those with custom glycosylation sites.
Characterize the glycan structures on your Fc fragments to understand their impact on function.
Reference
- Bates, A.; Power, C.A. David vs. Goliath: the structure, function, and clinical prospects of antibody fragments. Antibodies. 2019, 8(2): 28. (Open Access)
