Digestion of Mouse IgG2a and IgG3 Below the Hinge Region
CD BioGlyco provides high-quality Antibody Digestion Services for the digestion of mouse IgG2a and IgG3 below the hinge region research and ensures the continuity and transparency of the experimental process.
Digestion of Subclasses of Mouse IgG
Digestion of immunoglobulins has been widely used to prepare bivalent IgG antibody fragments lacking the Fc region. Studies have demonstrated that such F(ab')2 fragments can be used to analyze the role of Fc fragments in various biological functions. Digestive digestion has been applied to immunoglobulins from various species, including rabbits, humans, and mice.
With the development of hybridoma technology, the use of mouse antibodies with specificity and subclasses has greatly increased, and it has become particularly important to understand the effect of pepsin on the various subclasses of mouse IgG. In mice, the IgG class is divided into five subclasses (IgG1, IgG2a, IgG2b, IgG2c, and IgG3). It is worth mentioning that there is no general relationship between the subclasses of each species. However, enzymes that digest mouse IgG2a and IgG3 have yet to be developed.
Fig.1 Mouse IgG subclasses. (Temming, et al., 2020)
Key Technologies
Our service relies on a proprietary enzymatic digestion technology, engineered for unparalleled specificity and efficiency. While traditional proteases often exhibit broad activity that compromise the integrity of the desired fragments, our in-house developed enzyme is optimized to recognize and cleave the specific peptide sequence found below the hinge of mouse IgG2a and IgG3. This targeted approach ensures that the resulting Fc and F(ab')2 fragments are of the highest possible purity, retaining their native structure and biological function.
Digestion of Mouse IgG2a and IgG3 Below the Hinge Region at CD BioGlyco
CD BioGlyco provides a protease for the preparation of F(ab')2 from mouse subclasses IgG2a and IgG3. The protease is most effective at or near neutral pH and low NaCl concentrations. The protease is fast and easy to use and it digests mouse IgG2a and IgG3 at a specific site below the hinge region to generate intact F(ab')2 and Fc fragments. Since IgG is digested under mild conditions, its immunoreactivity is preserved.
We've crafted a straightforward and open-ended workflow, aiming to achieve peak efficiency and uphold top-notch quality throughout every stage of the project. Here's a detailed process:
- Project Consultation and Protocol Design
Our seasoned team of experts will engage in in-depth discussions with you, focusing on your project's objectives, the characteristics of your samples, and the specific requirements for antibody fragments. Based on these detailed exchanges and an understanding of your unique needs, we will meticulously design a personalized protocol. This protocol is tailored to optimize digestion parameters specifically for your mouse IgG2a or IgG3 antibodies, ensuring the best possible results.
- Sample Preparation and Quality Control
Once we receive your antibody samples, our dedicated team initiates a comprehensive evaluation process. We conduct stringent assessments of the sample's purity, concentration, and structural integrity. This meticulous quality control step is crucial to guarantee that the starting material meets all the necessary criteria for a successful digestion process.
- Enzymatic Digestion
The antibody samples are incubated with our exclusive enzyme under precisely defined conditions. Throughout the incubation period, the reaction is continuously monitored. Our goal is to ensure that the cleavage occurs completely and specifically beneath the hinge region, which results in the production of highly pure Fc and F(ab')2 fragments.
- Fragment Purification and Analysis
Following the digestion process, we utilize advanced chromatography techniques to isolate and purify the desired antibody fragments. To confirm the quality of the purified fragments, we employ multiple analytical methods. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) are used to verify the fragments' purity and structural integrity, ensuring that they meet the required standards.
- Data Reporting and Delivery
Finally, we provide you with a comprehensive final report. This report encompasses detailed information about the entire digestion process, in-depth analytical results, and clear chromatogram data. It offers a complete picture of the project's outcome, enabling you to fully understand the work that has been done and the quality of the results achieved.
Workflow
Publication Data
Journal: Molecular Immunology
IF: 3.0
Published: 2020
Results: This study comprehensively characterized the cross-reactivity of mouse IgG (mIgG) subclasses with human Fc gamma receptors (hFcγRs) using surface plasmon resonance imaging (SPRi), quantifying binding affinities for all mIgG subclasses to various hFcγRs. Key findings revealed that mIgG subclasses exhibit differential binding patterns, with mIgG2a/c and mIgG3 showing strong binding to hFcγRIa. mIgG1 displays high affinity for the hFcγRIIa-R131 variant, and no binding to hFcγRIIIb by any subclass, and that deglycosylation reduced most interactions but failed to eliminate binding of mIgG2a/c or mIgG3 to hFcγRIa or mIgG1 to hFcγRIIa-R131, except for mIgG2b. Therefore, this research provides critical insights for experimental design and identifying deglycosylated mIgG2b as a tool to minimize unwanted background binding in research.
Applications
- Therapeutic antibody development: The production of stable and functional Fc fragments is essential for engineering antibody-drug conjugates (ADCs) and bispecific antibodies.
- Glycosylation analysis: Precise digestion allows for the isolation of the Fc fragment, enabling detailed analysis of the glycosylation profile without interference from the Fab region.
- Immunological research: Separating the Fc and F(ab')2 fragments allows for independent study of their respective roles in immune cell activation, complement fixation, and antigen binding.
Advantages of Us
- One-stop service for digestion of mouse IgG2a and IgG3 below the hinge region
- Highly specific mouse IgG2a and IgG3 cleavage
- The proteases used do not require reducing agents or cofactors
- Responses allow the preservation of the immunoreactivity of IgG
- Comprehensive and reliable after-sales service
Frequently Asked Questions
CD BioGlyco has developed an advanced Glycoengineering Platform in glycoscience. Our professional research teams allow us to provide you with one-stop services for the digestion of mouse IgG2a and IgG3 below the hinge region. If you are interested in our services, please contact us for more details without any hesitation.
Associated Services
Reference
- Temming, A.R.; et al. Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding. Molecular Immunology. 2020, 127: 79-86. (Open Access)
