In antibody purification, Protein A and G chromatography methods have been established as indispensable for IgG purification. However, their limitations have become evident when applied to IgM. This article provides an overview of IgM purification processes, highlighting the procedures, advantages, and limitations associated with each method.
Pre-Treatment of IgM Samples
Natural IgM, like IgM expressed through hybridoma cells or recombinant methods, typically requires preliminary processing steps such as centrifugation and filtration to remove cellular debris and impurities. The processed sample can then be used in traditional IgM purification methods like ammonium sulfate precipitation and size exclusion chromatography. For affinity purification, additional steps vary. For example, thiophilic adsorption requires ammonium sulfate addition to facilitate antibody binding to the medium, while other affinity methods can proceed directly.
Ion exchange methods, however, require more complex preparation, including desalting and pH adjustment. This step is essential for optimal binding in ion exchange chromatography and can be achieved using dialysis or desalting columns, depending on sample needs and desired purity levels.
Common Methods for IgM Antibody Purification
Several traditional methods are commonly employed in IgM purification, including ammonium sulfate precipitation, size exclusion chromatography, ion exchange, and affinity chromatography. These methods vary in their approaches, making them suitable for different sample types and research applications.
1. Ammonium Sulfate Precipitation and Size Exclusion Chromatography
Ammonium sulfate precipitation and size exclusion chromatography are often combined for IgM purification. Initially, ammonium sulfate is used to selectively precipitate proteins based on their solubility in specific salt concentrations. This step helps in separating antibodies from unwanted proteins in the sample.
Following precipitation, size exclusion chromatography separates proteins by size. Given IgM's distinctively high molecular weight compared to other antibody classes, size exclusion effectively isolates it from smaller antibodies, ensuring higher purity.
2. Ion Exchange Chromatography
Ion exchange chromatography is frequently used for antibody purification, especially IgM, as it relies on the antibody's isoelectric point, typically ranging between 4-9. For IgM, cation exchange resins are often preferred to bind antibodies within the sample, providing high capacity and selectivity.
One of ion exchange chromatography's primary advantages is its ability to avoid residual host cell proteins (HCPs) and minimize ligand leaching. However, it lacks specificity, meaning it may not completely isolate IgM from other antibody types, making it less ideal when absolute IgM purity is required.
3. Affinity Chromatography
Affinity chromatography, a standard for IgG purification, is also used for IgM, leveraging specific ligands designed to bind IgM selectively. Various affinity ligands have been developed for IgM, each tailored to unique applications and offering distinct purification benefits.
3.1 Antibody-Based Affinity
In antibody-based affinity chromatography, anti-IgM antibodies are immobilized onto the chromatography medium. This approach can specifically capture IgM antibodies from complex mixtures. For example, anti-mouse IgM antibodies can be used to isolate mouse IgM, often utilized in immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) applications.
Another example is camelid single-domain antibodies, which can selectively purify human IgM without binding other immunoglobulin classes. These ligands offer high capacity, stability, and specificity, making them suitable for various IgM purification applications.
3.2 Antigen-Based Affinity
In antigen-based affinity chromatography, specific antigens act as ligands to capture IgM antibodies. A commonly used antigen is mannose-binding protein (MBP), also known as mannose-binding lectin (MBL), a serum protein from the mammalian liver. Covalently bonded to the chromatography medium, MBP serves as an affinity ligand for both human and murine IgM, though it exhibits lower binding capacity for human samples. This broadens IgM purification to multi-species applications, enhancing versatility.
3.3 Peptide Ligand Affinity
Peptide ligands specifically bind IgM, particularly synthetic peptides that mimic natural IgM-binding molecules. One example is a peptoid ligand with strong affinity, selectivity, and capacity (up to 15 mg/mL) for IgM. These peptide ligands are available for human, murine, and rat IgM purification, catering to species-specific research requirements.
3.4 Thiophilic Adsorption
Thiophilic adsorption is a dual-mode purification process utilizing a ligand's electron-donor and electron-acceptor properties. In this method, a 2-mercaptopyridine-based ligand interacts with IgM through its disulfide bonds. Due to its non-specific nature, thiophilic adsorption mainly applies to recombinant or hybridoma-derived IgM, as it shows limited efficacy with serum or ascites IgM.
3.5 Tag-Based Affinity
For recombinant IgM antibodies, affinity tags like His and FLAG tags can be incorporated at the gene-cloning stage. These small molecular tags allow efficient IgM purification with minimal structural or functional impact. Tag-based affinity provides high-purity IgM with reduced costs compared to conventional affinity ligands, and it is often used for high-throughput IgM production.
| Affinity Purification Method | Specificity | Capacity | Application Examples |
| Antibody-Based Affinity | High | Moderate | Immunoprecipitation, ChIP |
| Antigen-Based Affinity | High | Low | Multi-species IgM purification |
| Peptide Ligand Affinity | High | High | Human, murine, rat IgM |
| Thiophilic Adsorption | Low | Moderate | Recombinant IgM purification |
| Tag-Based Affinity | High | High | Recombinant IgM, high-throughput applications |
IgM antibodies are crucial in early infection diagnostics, often used in assays like ELISA and colloidal gold immunoassays for prompt detection. Due to IgM's antigen-binding and complement-activating abilities, it shows promise for therapeutic applications, providing advantages over IgG in some cases. As antibody therapeutics and in vitro diagnostics expand, demand for IgM purification services will grow.
With an extensive antibody platform and specialized team, Creative Biolabs offers advanced IgM production and purification services tailored to specific research requirements. Additional antibody services include IgA, IgE, and IgY expression and purification to meet diverse research and clinical needs. For inquiries, please reach out for a customized solution.
Recommended Services
| Services | Features | Price |
| IgM Production and Purification | Based on our comprehensive antibody platforms and professional experts, Creative Biolabs has absolute advantages in providing IgM production and purification services. | Inquiry |
| IgA Production and Purification | Our seasoned scientists can customize the most suitable IgA products and services according to the specific requirements of customers. | Inquiry |
| IgE Production and Purification | Our purification strategies utilize multiple methods, such as affinity chromatography, and size-exclusion chromatography, specifically designed to handle IgE's complex properties and ensure the highest purity. | Inquiry |
| IgY Production and Purification | Creative Biolabs has established a comprehensive platform offering a variety of non-IgG relevant services to global clients. IgY production and purification is one of what we are good at. | Inquiry |
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