MHC Class II, I-A Antibody

What is the specificity profile of the M5/114.15.2 monoclonal antibody?

The M5/114.15.2 monoclonal antibody recognizes mouse major histocompatibility complex class II molecules, binding to both I-A and I-E subregion-encoded glycoproteins. Specifically, it detects I-Ab, I-Ad, I-Aq, I-Ed, and I-Ek, but notably does not recognize I-Af, I-Ak, or I-As . This antibody detects a polymorphic determinant present on multiple immune cell types including B cells, monocytes, macrophages, dendritic cells, and activated T lymphocytes from mice carrying specific haplotypes (H-2b, H-2d, H-2q, H-2p, H-2r and H-2u) but not from mice with H-2s or H-2f haplotypes .

What research applications have been validated for the M5/114.15.2 antibody?

The M5/114.15.2 antibody has been validated for multiple research applications with specific parameters for each method:

For functional assays, particularly those requiring in vitro blocking of T cell responses, functional grade purified antibody preparations should be used . The antibody can be used at concentrations less than or equal to 0.125 μg per test for flow cytometric analysis of mouse splenocytes .

What are the key biological properties of MHC Class II molecules relevant to experimental design?

MHC Class II molecules are transmembrane glycoproteins expressed primarily on the surface of professional antigen-presenting cells. These molecules play a crucial role in the immune system by presenting extracellular peptides to CD4+ T-helper cells, orchestrating adaptive immune responses . When designing experiments involving MHC Class II, researchers should consider:

• Cell type specificity: MHC Class II is predominantly expressed on B cells, dendritic cells, and macrophages

• Functional role: Before surface presentation, MHC Class II molecules interact with endocytosed exogenous antigens

• Structural characteristics: The antigen-binding groove between MHC Class II alpha and beta chains is open at both ends and accommodates peptides 15-24 amino acids in length

• Expression regulation: Expression levels vary based on cell activation status and microenvironment

• Haplotype variations: Different mouse strains express distinct MHC Class II alleles with varying antibody reactivity

Understanding these properties is essential for accurately interpreting experimental results and designing controls for studies involving MHC Class II detection.

What protocol is recommended for optimizing M5/114.15.2 antibody in flow cytometry?

For optimal results in flow cytometry applications using the M5/114.15.2 antibody, researchers should implement a structured titration approach:

• Begin with the recommended concentration of ≤0.125 μg per test, where a test is defined as the amount of antibody needed to stain a cell sample in 100 μL final volume

• Prepare single-cell suspensions (typically mouse splenocytes) at consistent concentrations

• Create a dilution series of the antibody (e.g., 0.125, 0.06, 0.03, 0.015 μg per test)

• Include appropriate controls:

  • Isotype control (Rat IgG2b kappa)

  • Negative control (cells from H-2s or H-2f haplotype mice)

• Analyze by flow cytometry, evaluating both signal intensity and background staining

• Select the concentration providing maximum specific signal with minimal background

• Validate the selected concentration across different experimental conditions

Cell numbers should be determined empirically but can range from 10^5 to 10^8 cells per test . The antibody exhibits greater than 90% purity as determined by SDS-PAGE and less than 10% aggregation as measured by HPLC .

How should immunohistochemistry protocols be optimized for detecting MHC Class II in different tissue preparations?

Optimizing MHC Class II detection requires different approaches for frozen versus paraffin-embedded tissues:

For frozen sections:

• Fixation: Use 2-4% paraformaldehyde briefly to preserve epitope accessibility

• Blocking: Employ species-specific serum with detergent to reduce background

• Antibody dilution: Start with 1:50-1:200 dilution and optimize

• Incubation: Overnight at 4°C typically yields optimal signal-to-noise ratio

• Detection system: Fluorescent secondary antibodies or amplification systems as needed

For paraffin sections:

• Antigen retrieval: Critical step - typically requires heat-induced epitope retrieval

• Peroxidase blocking: Treatment with hydrogen peroxide to reduce endogenous peroxidase activity

• Antibody concentration: Higher concentrations often required compared to frozen sections

• Incubation time: Extended incubation periods may be necessary for optimal staining

• Signal amplification: Consider using signal enhancement systems for weaker signals

For both methods, validation with appropriate positive controls (lymphoid tissues) and negative controls (tissues from non-reactive haplotypes) is essential for confirming specificity .

What factors influence the efficacy of M5/114.15.2 in functional blocking assays?

The M5/114.15.2 antibody exhibits haplotype-specific inhibition of I-A-restricted T cell responses, which influences its efficacy in functional blocking assays. Key factors include:

• Haplotype dependence: The antibody inhibits responses in H-2b, H-2d, H-2q, and H-2u haplotypes but not in H-2f, H-2k, or H-2s haplotypes

• Antibody concentration: Optimal blocking concentration must be determined empirically through dose-response experiments

• Antibody format: For functional blocking assays, use functional grade antibody preparations to ensure appropriate activity and minimal contamination

• Target cell type: Efficacy may vary based on the specific antigen-presenting cell population

• Experimental timing: Adding the antibody at different stages of T cell activation can affect blocking efficacy

• Readout selection: Choose appropriate functional readouts (proliferation, cytokine production) to accurately assess blocking activity

Researchers should include appropriate positive and negative control haplotypes when evaluating blocking efficacy, and consider that different experimental systems may require different antibody concentrations for optimal inhibition.

How can MHC Class II antibodies be leveraged in cancer immunotherapy research?

MHC Class II antibodies provide valuable tools for cancer immunotherapy research through several approaches:

• Tumor microenvironment characterization: Recent studies show that prostate tumors from both human patients and mouse models express MHC Class I on tumor epithelial cells and MHC Class II on hematopoietic lineage cells within the tumor . This expression pattern suggests a permissive environment for T-cell-mediated immunotherapeutic approaches .

• Biomarker development: MHC Class II expression on tumor-infiltrating immune cells may serve as a predictive biomarker for immunotherapy response. Standardized immunohistochemistry protocols using M5/114.15.2 can quantify this expression.

• Therapeutic targeting: Engineered anti-MHC II antibodies can potentially enhance antigen presentation and T cell activation within the tumor microenvironment.

• Response monitoring: Using M5/114.15.2 in flow cytometry panels can track changes in MHC Class II expression on tumor-infiltrating immune cells during immunotherapy treatment.

• Combination therapy design: Understanding the expression patterns of MHC molecules informs rational design of combination immunotherapies targeting both tumor cells and the tumor microenvironment.

The observation that prostate tumors show significant CD3+ T cell infiltration alongside MHC expression further supports the potential of MHC-targeted approaches in cancer immunotherapy research.

What techniques can address cross-reactivity challenges when using M5/114.15.2 in mixed haplotype systems?

Working with M5/114.15.2 antibody in mixed haplotype experimental systems requires specialized approaches:

• Haplotype-specific validation:

  • Test the antibody on cells from mice with known haplotypes

  • Establish baseline reactivity patterns for different haplotypes

  • Generate reference data for each haplotype to guide interpretation

• Competitive binding assay:

  • Mix cells from reactive and non-reactive haplotypes at defined ratios

  • Perform antibody titration to identify concentrations maintaining specificity

  • Calculate specificity indices comparing signal between reactive and non-reactive haplotypes

• Complementary antibody approach:

  • For comprehensive coverage, use additional antibodies specific for haplotypes not recognized by M5/114.15.2

  • Create a panel approach ensuring detection across all relevant haplotypes

  • Normalize signals using appropriate standards for inter-experimental comparison

• Genetic background considerations:

  • For chimeric models, implement parallel staining with haplotype-specific markers

  • Use bioinformatic approaches to deconvolute mixed populations

  • Apply clustering methods to separate populations by reactivity profiles

For experimental systems containing H-2s or H-2f haplotypes, researchers should be particularly cautious as M5/114.15.2 does not recognize MHC Class II from these genetic backgrounds .

How do post-translational modifications of MHC Class II affect antibody detection efficiency?

Post-translational modifications (PTMs) of MHC Class II molecules can significantly impact antibody recognition and experimental outcomes:

• Glycosylation effects:

  • N-linked glycosylation sites on MHC II can sterically hinder antibody access

  • Compare native versus enzymatically deglycosylated samples to assess impact

  • Consider modification-specific detection methods for comprehensive analysis

• Protein processing:

  • MHC Class II molecules undergo processing including the removal of invariant chain

  • Different antibody clones may preferentially recognize mature versus immature forms

  • Include time-course experiments to capture different stages of processing

• Conformational changes:

  • Peptide loading alters MHC II conformation, potentially affecting epitope accessibility

  • Compare empty versus peptide-loaded MHC II molecules when evaluating antibody binding

  • Consider native versus denatured conditions in applications like Western blotting

• Experimental condition effects:

  • Fixation protocols may alter epitope accessibility through protein cross-linking

  • Detergent selection in lysis buffers affects membrane protein solubilization and epitope preservation

  • Sample handling (freeze-thaw cycles, storage conditions) can impact modification status

Researchers should implement specific controls to account for these PTM effects when designing experiments involving MHC Class II detection and functional studies.

What considerations are critical for co-immunoprecipitation studies using anti-MHC Class II antibodies?

Co-immunoprecipitation (co-IP) studies with anti-MHC Class II antibodies require careful methodological attention:

• Buffer optimization:

  • Use mild non-ionic detergents (NP-40 or Triton X-100) to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation of associated peptides and proteins

  • Consider digitonin for membrane protein complexes when studying the peptide-loading complex

• Antibody coupling strategy:

  • Direct coupling to beads reduces heavy/light chain interference in Western blotting

  • Pre-clear lysates with isotype control antibodies to minimize non-specific binding

  • Consider crosslinking antibodies to beads to prevent co-elution during analysis

• Precipitation conditions:

  • Longer incubation times improve yield for lower-abundance complexes

  • Gentler washing conditions preserve weaker interactions in the MHC II complexes

  • Consider sequential IPs to enrich for specific subcomplexes

• Experimental validation:

  • Include isotype-matched control antibodies to identify non-specific binding

  • Use cells from non-reactive haplotypes (H-2s or H-2f) as negative controls

  • Perform reciprocal IPs where possible to confirm interactions

These methodological considerations help ensure that co-IP results accurately reflect the in vivo interactions of MHC Class II molecules rather than experimental artifacts.

How can single-cell analysis with MHC Class II antibodies reveal immune cell heterogeneity?

Single-cell analysis using MHC Class II antibodies provides powerful insights into the heterogeneity of antigen-presenting cells through several methodological approaches:

• Multi-parameter flow cytometry:

  • Use M5/114.15.2 alongside lineage markers (CD11c, F4/80, CD19)

  • Add activation markers to correlate MHC II expression with functional state

  • Implement viability dye to exclude false positive signals from dead cells

  • Apply quantitative analysis of expression levels beyond positive/negative classification

• Mass cytometry integration:

  • Metal-conjugated M5/114.15.2 enables simultaneous detection of multiple parameters

  • Pair with transcription factor staining to correlate with regulatory mechanisms

  • Implement dimensionality reduction techniques to identify novel MHC II-expressing subpopulations

• Spatial analysis methodologies:

  • Multiplex immunofluorescence with M5/114.15.2 and other immune markers

  • Quantify spatial relationships between MHC II+ cells and T cells in tissue contexts

  • Analyze neighborhood interactions to understand functional organization

This multi-faceted approach reveals not only which cells express MHC Class II but also their functional state, spatial organization, and relationship to other immune cells within complex tissues such as tumor microenvironments .

What are the considerations for quantitative analysis of MHC Class II expression?

Quantitative analysis of MHC Class II expression requires attention to several methodological details:

• Standardization approaches:

  • Use quantitative beads with known antibody binding capacity for absolute quantification

  • Calculate antibodies bound per cell rather than relying on relative mean fluorescence intensity

  • Include standardized cells across experiments for inter-experimental normalization

• Control implementation:

  • Fluorescence-minus-one controls to set positive/negative boundaries

  • Isotype control (Rat IgG2b) to assess non-specific binding

  • Biological controls: Include cells from non-reactive haplotypes (H-2s or H-2f) as negative controls

• Sample preparation variables:

  • Cell isolation method impacts MHC II detection (enzymatic digestion can cleave epitopes)

  • Fixation affects epitope accessibility (concentrations above 2% can reduce detection)

  • Temperature effects: Perform staining at 4°C to prevent internalization

• Advanced analysis approaches:

  • Use appropriate visualization methods for complex expression patterns

  • Consider density estimation for heterogeneous populations

  • Implement clustering algorithms for unbiased population identification

This structured approach ensures reproducible and accurate quantification of MHC Class II expression across experimental conditions and genetic backgrounds.

What future research directions are emerging for MHC Class II antibodies?

The field of MHC Class II antibody research continues to evolve with several promising directions:

• Increased integration with single-cell technologies to reveal functional heterogeneity of antigen-presenting cells

• Development of more specific antibodies against different MHC Class II allelic variants to enable more precise haplotype-specific studies

• Application in cancer immunotherapy research, leveraging the finding that MHC Class II molecules are expressed in tumor microenvironments

• Advanced imaging applications combining MHC Class II detection with spatial transcriptomics

• Therapeutic applications targeting the MHC Class II presentation pathway to modulate immune responses

The M5/114.15.2 antibody remains a valuable tool for these emerging research directions, with its well-characterized specificity profile and validated applications across multiple experimental systems .