HLA-DOB Antibody

What is HLA-DOB and what is its functional role in immune processes?

HLA-DOB is a protein that forms part of the major histocompatibility complex (MHC) class II family. It functions as an important modulator in the HLA class II restricted antigen presentation pathway by interacting with the HLA-DM molecule in B-cells . Specifically, HLA-DOB modifies the peptide exchange activity of HLA-DM, which is crucial for antigen presentation to CD4+ T cells .

The canonical protein has 273 amino acid residues with a molecular mass of approximately 30.8 kDa . At the subcellular level, HLA-DOB is primarily localized in endosomes and lysosomes, consistent with its role in antigen processing and presentation . This protein is particularly important in the context of autoimmune diseases, as variations in genes encoding these molecules have been linked to susceptibility to conditions such as rheumatoid arthritis .

What tissues and cell types express HLA-DOB?

HLA-DOB shows notable expression in specific tissues and cell types:

This expression pattern has been demonstrated in immunohistochemistry studies using anti-HLA-DOB antibodies, where human tonsil tissue shows strong positive staining while tissues like skeletal muscle exhibit no detectable expression . At the cellular level, B-cell lines like Raji and Ramos express HLA-DOB, whereas T-cell lines such as MOLT-4 typically show minimal expression .

What are the common applications and validated protocols for HLA-DOB antibodies?

HLA-DOB antibodies have been validated for multiple research applications:

When selecting an antibody, consider the intended application, species reactivity (most are human-specific), and preferred detection method. For example, DOB.L1 clone (a mouse monoclonal IgG2b) has been extensively validated for Western blot and IHC-P applications with human samples .

What post-translational modifications (PTMs) occur on HLA-DOB and how do they affect detection?

Several important post-translational modifications affect HLA-DOB:

These modifications can impact experimental outcomes. For Western blotting, glycosylation causes the observed molecular weight to be higher than the 30.8 kDa predicted from the amino acid sequence alone . This can be confirmed by treating samples with glycosidases like PNGase F, which removes N-linked glycans and results in a downward shift in band migration . When interpreting results, it's important to consider how these PTMs might affect antibody binding and apparent molecular weight.

How should researchers select positive and negative controls for HLA-DOB studies?

Proper controls are essential for validating HLA-DOB antibody specificity:

Recommended Positive Controls:

• Cell lines: Raji and Ramos (human Burkitt's lymphoma B lymphocytes)

• Tissues: Human tonsil, spleen, lymph nodes

Recommended Negative Controls:

• Cell lines: MOLT-4 (human T lymphoblasts)

• Tissues: Human skeletal muscle

Technical Control Procedures:

• Secondary antibody only: Omit primary antibody to assess background

• Isotype control: Use matched isotype (mouse IgG2b for DOB.L1 clone)

• Peptide competition: Pre-incubate antibody with immunizing peptide

• For glycosylation studies: Compare untreated vs. PNGase F-treated samples

Each experiment should include both positive and negative biological controls along with appropriate technical controls to ensure valid interpretation of results.

How does the interaction between HLA-DOB and HLA-DM regulate antigen presentation?

HLA-DOB, together with HLA-DOA, forms the HLA-DO heterodimer that serves as a key modulator in the MHC class II antigen presentation pathway. This molecular interaction occurs through several mechanisms:

• Peptide Exchange Modulation: HLA-DO interacts directly with HLA-DM to modify its peptide exchange activity . This interaction affects which peptides are ultimately loaded onto MHC class II molecules.

• pH-Dependent Regulation: The interaction between HLA-DO and HLA-DM is highly pH-sensitive, with maximum inhibitory activity occurring at endosomal pH ranges.

• B-cell Specific Modulation: While HLA-DM is expressed in various antigen-presenting cells, HLA-DO expression is more restricted, particularly in B cells, creating cell type-specific regulation of antigen presentation .

When studying this interaction, researchers should consider:

• The subcellular localization (endosomes and lysosomes)

• The potential impact of post-translational modifications on protein-protein interactions

• The differential expression patterns in various cell types and tissues

This molecular mechanism helps fine-tune the repertoire of peptides presented to CD4+ T cells, thereby influencing adaptive immune responses and potentially contributing to autoimmune disease susceptibility .

What is the relationship between Class II Transactivator (CIITA) and HLA-DOB expression?

The regulation of HLA-DOB expression differs from classical MHC class II genes in its relationship with CIITA (Class II Transactivator):

This regulatory pattern has important implications for researchers:

• When studying HLA-DOB in different cell systems, CIITA status should be considered

• Experimental manipulation of CIITA can be used to modulate HLA-DOB expression

• Comparing HLA-DOB with classical MHC II genes in the same samples can provide insights into differential regulation

Understanding this relationship provides insights into the unique regulatory mechanisms controlling HLA-DOB expression and its role in antigen presentation.

How can researchers accurately distinguish between specific and non-specific signals when detecting HLA-DOB?

Distinguishing specific from non-specific signals is critical for accurate HLA-DOB detection. Advanced approaches include:

• Glycosylation Analysis for Western Blot:

  • Untreated samples show bands at ~30 kDa (glycosylated form)

  • PNGase F-treated samples shift to lower molecular weight

  • This mobility shift confirms glycoprotein identity and antibody specificity

• Multiple Detection Methods Consensus:

  • Confirm findings across different techniques (WB, IHC, FCM)

  • Compare results with different antibody clones targeting distinct epitopes

• Genetic Validation Approaches:

  • siRNA or CRISPR-based knockdown/knockout of HLA-DOB

  • Heterologous expression systems with controlled HLA-DOB expression

• Critical Control Samples:

  • Known positive B cell lines (e.g., Raji, Ramos) versus negative T cell lines (e.g., MOLT-4)

  • Side-by-side comparison of positive tissues (tonsil) with negative tissues (skeletal muscle)

• Signal Quantification:

  • Establish signal-to-noise ratios across different antibody concentrations

  • Implement digital image analysis with appropriate thresholding

These approaches collectively minimize false positives and increase confidence in specific HLA-DOB detection across experimental platforms.

What experimental considerations are important when studying HLA-DOB allelic variants?

The HLA-DOB gene has 13 allelic variants reported in the Allele Frequency Net Database , presenting several important considerations:

• Antibody Epitope Mapping:

  • Determine whether commercial antibodies target conserved or polymorphic regions

  • Epitopes in conserved regions detect all alleles; those in polymorphic regions may be allele-specific

• Population Genetics Considerations:

  • Different populations exhibit varying HLA-DOB allele frequencies

  • Study design should account for potential population stratification

• Validation Across Allelic Variants:

  • When possible, test antibody reactivity with cells expressing different HLA-DOB alleles

  • Consider using multiple antibodies targeting different epitopes for comprehensive detection

• Functional Impact of Polymorphisms:

  • Assess whether allelic variations affect protein-protein interactions, particularly with HLA-DM

  • Consider potential influence on post-translational modifications

Understanding these considerations is particularly important for immunogenetic studies and transplantation research where allelic variation may have functional significance.

How should researchers design experiments to study HLA-DOB in the context of donor-specific antibodies in transplantation?

Recent transplantation research indicates that donor-specific antibodies (DSAs) targeting various HLA loci, including HLA-DOB, can develop after transplantation . When designing experiments in this context:

• Differentiate True vs. False Positive Reactions:

  • True donor-specific HLA-DOB antibodies must be distinguished from false positives

  • Implement appropriate controls and thresholds for mean fluorescence intensity (MFI)

  • Consider that HLA-DQ is more frequently targeted (54.2%) than other loci

• Comprehensive HLA Loci Analysis:

  • Include HLA-DOB alongside more commonly studied loci

  • Data show dnDSA target distribution varies significantly (HLA-DQ: 54.2%, HLA-DP: 4.6%, HLA-C: lower frequency)

• Epitope Analysis Approaches:

  • Implement PIRCHE-II scoring or similar methods to predict immunogenicity

  • Recent data shows significant differences in PIRCHE II scores for true vs. false dnDSA directed at HLA Class II antigens

• Clinical Correlation Studies:

  • Design studies to correlate HLA-DOB antibody development with clinical outcomes

  • Track rejection episodes, graft function parameters, and long-term survival

This methodological framework enables researchers to comprehensively assess the clinical significance of HLA-DOB antibodies in transplantation contexts.

What methodological approaches can researchers use to study HLA-DOB interactions with other components of the antigen presentation machinery?

Studying HLA-DOB's interactions with other components of the antigen presentation machinery requires sophisticated methodological approaches:

• Co-immunoprecipitation Protocols:

  • Use anti-HLA-DOB antibodies to pull down protein complexes

  • Available agarose-conjugated antibodies facilitate this approach

  • Analyze co-precipitated proteins (particularly HLA-DM) by Western blot or mass spectrometry

• FRET/BRET Analysis:

  • Tag HLA-DOB and interaction partners with appropriate fluorophores/bioluminescent proteins

  • Measure energy transfer as indicator of protein proximity and interaction

• Proximity Ligation Assays:

  • Detect protein-protein interactions at endogenous expression levels

  • Particularly useful for visualizing interactions in subcellular compartments

• Functional Peptide Loading Assays:

  • Measure impact of HLA-DOB on peptide loading onto MHC class II molecules

  • Compare wild-type cells with those having modified HLA-DOB expression

• Super-resolution Microscopy:

  • Visualize co-localization of HLA-DOB with other components in endosomal/lysosomal compartments

  • Available fluorophore-conjugated antibodies (FITC, PE, Alexa Fluor conjugates) facilitate this approach

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Map interaction interfaces between HLA-DOB and binding partners

  • Identify structural changes upon complex formation

These methodological approaches provide comprehensive insights into HLA-DOB's molecular interactions and functional role in the antigen presentation pathway.

How can researchers effectively study the impact of post-translational modifications on HLA-DOB function?

Investigating the functional impact of HLA-DOB post-translational modifications requires specialized approaches:

• Site-directed Mutagenesis Strategy:

  • Create point mutations at key modification sites:

    • N45 (N-glycosylation)

    • Y63, S68, S97 (phosphorylation)

    • C105 (methylation)

  • Express mutant proteins and assess functional consequences

• Phosphorylation-specific Detection:

  • Use phospho-specific antibodies if available

  • Implement phosphatase treatments as controls

  • Consider phosphomimetic mutations (E/D substitutions) for functional studies

• Mass Spectrometry-based PTM Mapping:

  • Identify and quantify specific modifications

  • Compare PTM profiles between different cell types or activation states

  • Correlate PTM changes with functional outcomes

• Structure-Function Analysis:

  • Model impact of PTMs on protein structure

  • Assess how modifications affect interaction surfaces with HLA-DM

These methodological approaches provide a comprehensive framework for understanding how post-translational modifications regulate HLA-DOB function in antigen presentation pathways.