MS4A1 Monoclonal Antibody

What is MS4A1 and what is its basic structure?

MS4A1 (membrane spanning 4-domains A1), commonly known as CD20, is a member of the membrane-spanning 4A gene family. In humans, the canonical protein consists of 297 amino acid residues with a molecular mass of approximately 33.1 kDa . As its name suggests, MS4A1 is characterized by four transmembrane domains with both the N- and C-termini located inside the cell. The protein is primarily localized in the cell membrane and has up to two different isoforms reported in humans . The gene encoding MS4A1 is located on chromosome 11q12, among a cluster of related family members . Alternative splicing of this gene results in two transcript variants that encode the same protein .

What cellular functions does MS4A1/CD20 perform?

MS4A1/CD20 serves as a B-lymphocyte surface molecule that plays a crucial role in the development and differentiation of B-cells into plasma cells . It is involved in B-cell differentiation and receptor-mediated signaling pathways . The protein undergoes post-translational modifications, including phosphorylation, which likely regulate its function . Recent groundbreaking research has expanded our understanding of MS4A1 function beyond the immune system, revealing that it also functions as an olfactory receptor in mice that recognizes compounds produced by predators, triggering innate avoidance behaviors critical for survival . This dual functionality makes MS4A1 a particularly interesting target for interdisciplinary research spanning immunology and neuroscience.

What is the expression pattern of MS4A1/CD20?

MS4A1/CD20 is predominantly expressed on B-cells in the immune system . More specifically, it serves as a marker for identifying various B-cell subpopulations including immature B cells, memory B cells, naïve B cells, large intestine lamina propria lymphocytes, and plasma cells . Remarkably, recent research has identified MS4A1 expression in a previously uncharacterized subpopulation of olfactory sensory neurons (OSNs) in the main olfactory epithelium of the murine nasal cavity . These MS4A1-expressing neurons have cell bodies situated in the same anatomic location as OSN cell bodies, extend sensory dendrites to the lumen of the olfactory epithelium, and project axonal-like structures toward the olfactory bulb . Co-staining experiments have confirmed that these MS4A1-expressing cells are indeed neurons, as they express the neuronal marker NeuN .

How are MS4A1 recombinant monoclonal antibodies produced?

The production of MS4A1 recombinant monoclonal antibodies follows a sophisticated multi-step process designed to ensure exceptional quality and specificity. The process begins with isolating B cells from an animal immunized with recombinant human MS4A1 protein. Total RNA is extracted from these B cells and converted to cDNA through reverse transcription. MS4A1 antibody genes are then amplified using primers designed for antibody constant regions and inserted into an expression vector . This vector is subsequently transfected into host cells to facilitate antibody production. Following cell culture, the antibody is harvested from the supernatant and purified using affinity chromatography, resulting in a highly purified product suitable for various research applications . Alternatively, some MS4A1 monoclonal antibodies are purified from mouse ascites fluids or tissue culture supernatant by protein A/G affinity chromatography .

How is the specificity of MS4A1 monoclonal antibodies validated?

Validation of MS4A1 monoclonal antibodies typically involves multiple complementary approaches to ensure specificity. ELISA is commonly conducted to validate an antibody's specificity and functionality in detecting human MS4A1 protein . More rigorous validation includes testing on MS4A1 transfectants versus irrelevant transfectants to confirm specific binding . For antibodies intended for immunohistochemistry and in vivo applications, cross-validation using multiple antibodies recognizing different epitopes is essential. In one study, three different anti-MS4A1 antibodies (raised in different species and recognizing different MS4A1 epitopes) were found to co-label the same cells in the mouse olfactory epithelium, confirming specificity . Additionally, these antibodies did not stain any cells in olfactory epithelial sections obtained from MS4A1 knockout mice, further confirming their specificity . For flow cytometry applications, validation often includes co-staining with established B-cell markers such as CD19 .

What are the optimal conditions for using MS4A1 antibodies in flow cytometry?

Flow cytometry is one of the most common applications for MS4A1/CD20 antibodies, particularly for identifying and sorting B-cell populations. For optimal results, researchers should consider several key parameters. MS4A1 antibodies conjugated to fluorophores such as APC (allophycocyanin) are particularly effective for flow cytometry applications . When designing flow cytometry panels, MS4A1 antibodies can be effectively paired with other B-cell markers such as CD19 for comprehensive B-cell phenotyping, as demonstrated in validation studies .

For staining membrane-associated proteins like MS4A1:

• Use freshly isolated cells whenever possible

• Maintain appropriate cell concentrations (typically 1×10^6 cells/100 μL)

• Incubate with antibody at proper dilution (follow manufacturer's recommendations, typically starting at 1:100)

• Include proper isotype controls (e.g., Mouse IgG1 APC for MS4A1 APC-conjugated antibodies)

• Perform compensation when using multiple fluorophores

For MS4A1 detection in peripheral blood lymphocytes, co-staining with CD19 PE-conjugated antibodies allows for accurate identification of B-cell populations expressing MS4A1 . Additionally, optimizing fixation protocols is critical if intracellular staining is required alongside MS4A1 detection.

How should MS4A1 antibodies be optimized for Western blot applications?

For Western blot applications, MS4A1 antibodies require careful optimization to detect the approximately 33 kDa protein effectively. Based on manufacturer recommendations, the following protocol optimizations are suggested:

• Sample preparation:

  • Include proper membrane protein extraction techniques as MS4A1 is a membrane protein

  • Use appropriate detergents (e.g., RIPA buffer with 1% NP-40 or Triton X-100)

  • Consider non-reducing conditions as reducing agents may alter epitope recognition

• Antibody dilution:

  • Start with the recommended 1:500 dilution

  • Perform titration experiments (1:250 to 1:1000) to determine optimal signal-to-noise ratio

• Blocking and incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Use secondary antibodies appropriate for the host species (anti-mouse for mouse monoclonal antibodies)

• Detection considerations:

  • Enhanced chemiluminescence (ECL) substrates are typically effective

  • For low expression targets, consider signal amplification systems

MS4A1 antibodies used for Western blot applications should be validated against positive control samples (B-cell lines) and negative control samples (non-B-cell lines) to confirm specificity.

What are the critical factors for successful immunohistochemistry with MS4A1 antibodies?

Immunohistochemistry (IHC) with MS4A1 antibodies requires attention to several critical factors for optimal staining, particularly given its membrane localization:

• Tissue preparation and antigen retrieval:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended

  • Optimal section thickness is typically 4-6 μm

• Antibody concentration and incubation:

  • Start with the recommended 1:100 dilution

  • Incubate at 4°C overnight or room temperature for 1-2 hours

  • Include proper isotype controls

• Detection systems:

  • Polymer-based detection systems often provide superior sensitivity

  • For dual or multiplex staining, use antibodies from different host species or directly conjugated antibodies

• Controls and validation:

  • Include positive control tissues (lymphoid tissues, tonsil)

  • Use negative control tissues (tissues lacking B cells)

  • Consider dual staining with other B-cell markers for confirmation

• Interpretation considerations:

  • MS4A1/CD20 exhibits characteristic membranous staining pattern in B cells

  • Assess background staining in non-B cell areas to ensure specificity

For non-lymphoid tissues, such as the olfactory epithelium, modified protocols may be necessary. In studies examining MS4A1 expression in olfactory sensory neurons, co-staining with neuronal markers like NeuN helped confirm the identity of MS4A1-positive cells .

How can MS4A1 knockout models advance our understanding of MS4A1 function?

MS4A1 knockout models have provided critical insights into the multifaceted functions of this protein beyond its established role in B-cell biology. Recent research utilizing MS4A1-deficient mice has revealed that MS4A1 functions as an olfactory receptor mediating innate avoidance behaviors in response to predator-derived compounds . When exposed to 2,5-dimethylpyrazine (2,5-DMP), a compound found in predator urine, wildtype mice exhibited strong avoidance behaviors. In contrast, MS4A1 knockout mice displayed no avoidance responses to this predator-derived compound, demonstrating the essential role of MS4A1 in mediating this specific behavioral response .

Importantly, the behavioral deficits in MS4A1 knockout mice were highly specific to 2,5-DMP detection. The knockout mice retained normal avoidance responses to other aversive odorants such as TMT (2,4,5-trimethylthiazoline), exhibited similar locomotive behaviors to wildtype mice, and performed normally in anxiety assays such as the elevated plus maze . This specificity indicates that MS4A1 functions as a dedicated olfactory receptor for specific compounds rather than affecting general olfactory processing or anxiety-related behaviors.

To leverage MS4A1 knockout models effectively, researchers should:

• Include appropriate behavioral control assays to distinguish specific MS4A1-dependent phenotypes from general deficits

• Perform comprehensive histological analysis to assess effects on both the immune system and olfactory system

• Consider potential compensatory mechanisms that may emerge in constitutive knockout models

• Use tissue-specific conditional knockout approaches to disambiguate the roles of MS4A1 in different systems

What emerging roles of MS4A1 beyond B-cell biology should researchers investigate?

The recent discovery of MS4A1 as an olfactory receptor in mice opens exciting new research directions beyond traditional B-cell biology. Researchers should consider investigating:

• Sensory neuron function: MS4A1 is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium . These neurons extend sensory dendrites to the lumen of the olfactory epithelium and project axonal-like structures toward the olfactory bulb. Future research should map the precise neural circuits involving these MS4A1-expressing neurons and determine how they integrate with the broader olfactory system.

• Innate behavior modulation: MS4A1 mediates innate avoidance responses to specific predator-derived compounds such as 2,5-dimethylpyrazine (2,5-DMP) . This suggests MS4A1 may be part of a specialized olfactory subsystem dedicated to detecting ethologically relevant odors that trigger unlearned behaviors. Researchers should investigate the full spectrum of odors detected by MS4A1 and the behavioral outputs they trigger.

• Evolutionary conservation: Given the dual role of MS4A1 in both immune and olfactory systems, comparative studies across species could reveal how this functional duality evolved. In particular, determining whether the olfactory function of MS4A1 is conserved in humans would have significant implications.

• Cross-talk between systems: The expression of a canonical immune protein in the olfactory system raises questions about potential cross-talk between immune and nervous systems. Researchers should investigate whether inflammatory responses modulate MS4A1 function in olfactory neurons and, conversely, whether olfactory stimuli might affect MS4A1 function in B-cells.

• Structural basis of ligand detection: Crystal structures or cryo-EM studies of MS4A1 bound to its ligands could reveal how a protein evolved to recognize both B-cell signaling partners and specific odorants.

How can combined genetic and antibody-based approaches advance MS4A1 research?

Integrating genetic and antibody-based approaches creates powerful research paradigms that can provide comprehensive insights into MS4A1 function across biological systems:

• Validation of antibody specificity: Genetic models provide the gold standard for antibody validation. As demonstrated in recent research, anti-MS4A1 antibodies showed no staining in olfactory epithelial sections from MS4A1 knockout mice, confirming their specificity . This combined approach ensures that observed signals truly represent MS4A1 expression rather than cross-reactivity.

• Structure-function correlation: Site-directed mutagenesis of MS4A1 coupled with antibodies recognizing specific domains can elucidate which regions of the protein are critical for different functions. For example, antibodies targeting different epitopes could be used to determine which domains are essential for odorant binding versus immune signaling.

• Cell-type specific manipulation: Conditional knockout strategies can eliminate MS4A1 expression in specific cell types (e.g., B cells versus olfactory neurons), while antibodies can be used to track the consequences of these manipulations across tissues. This approach can disambiguate the cell-autonomous versus non-cell-autonomous effects of MS4A1 deletion.

• Temporal regulation studies: Inducible genetic systems allow temporal control over MS4A1 expression, while antibodies provide rapid detection methods to confirm protein depletion or restoration. This combination is particularly valuable for developmental studies examining when MS4A1 expression is critical for proper system function.

• In vivo tracking and manipulation: Genetic approaches like knock-in fluorescent reporters can be combined with antibody-based cell sorting to isolate specific MS4A1-expressing cell populations for transcriptomic or proteomic analysis. Additionally, antibody-drug conjugates can be used for targeted manipulation of MS4A1-expressing cells in genetically modified backgrounds.

How should MS4A1 monoclonal antibodies be stored and handled for optimal performance?

Proper storage and handling of MS4A1 monoclonal antibodies are crucial for maintaining their performance and extending their usable lifespan. Based on manufacturer recommendations and best practices:

• Storage conditions:

  • Store lyophilized antibodies at -20°C as received

  • Once reconstituted, aliquot antibodies to avoid repeated freeze-thaw cycles

  • For long-term storage of reconstituted antibodies, store at -20°C or -80°C

  • For short-term storage (1-2 weeks), 4°C is typically acceptable

• Reconstitution protocols:

  • For lyophilized antibodies, add the recommended volume of distilled water to achieve the desired concentration (typically 1 mg/mL)

  • Allow the lyophilized product to completely dissolve before use

  • For carrier-free antibodies intended for conjugation, perform an additional desalting process using appropriate desalting columns (e.g., Zeba Spin Desalting Columns, 7KMWCO)

• Stability considerations:

  • Most MS4A1 antibodies remain stable for 12 months from the date of receipt when stored properly

  • Avoid exposure to direct light, particularly for fluorophore-conjugated antibodies

  • Maintain sterile conditions when handling to prevent microbial contamination

• Transport and shipping:

  • Most MS4A1 antibodies can be shipped at ambient temperature

  • Upon receipt, transfer to recommended storage conditions immediately

• Quality control:

  • Before using in critical experiments, validate antibody performance with positive controls

  • Consider periodic validation testing for antibodies stored long-term

What are the common challenges when using MS4A1 antibodies and how can they be addressed?

What are the considerations for selecting the optimal MS4A1 antibody clone for specific research applications?

Selecting the appropriate MS4A1 antibody clone is crucial for experimental success and requires consideration of several application-specific factors:

• Epitope specificity:

  • Different MS4A1 clones recognize distinct epitopes, which may be differentially accessible in various applications

  • For studying specific MS4A1 domains, select clones with mapped epitopes in regions of interest

  • Consider using multiple antibodies recognizing different epitopes for cross-validation

• Application suitability:

  • Western Blot: Clones that recognize linear epitopes (e.g., UMAB38) are typically more effective

  • Flow Cytometry: Clones recognizing extracellular domains (e.g., clone 396444) are preferred

  • IHC: Clones validated specifically for IHC applications (e.g., 2F4 clone) offer better results

  • Multiple applications: Some versatile clones (e.g., UMAB38) work across multiple applications

• Species cross-reactivity:

  • Most MS4A1 antibodies are specific to a single species

  • Some clones demonstrate cross-reactivity with MS4A1 from multiple species, which is valuable for comparative studies

  • The 2F4 clone shows reactivity with human, mouse, and rat MS4A1 , making it suitable for cross-species studies

• Host species considerations:

  • Consider the host species (typically mouse for MS4A1 monoclonal antibodies ) when designing multi-color flow panels or multiplex IHC

  • Avoid using secondary antibodies that might cross-react with endogenous immunoglobulins in your sample

• Validation evidence:

  • Review the validation data provided by manufacturers

  • Look for antibodies validated in knockout systems

  • Consider antibodies validated using high-density protein chips or other specificity tests

  • For novel applications (e.g., olfactory research), select antibodies validated in relevant tissues