BOB1 Antibody

What is BOB1 and what is its biological significance?

BOB1 (also known as OBF-1, OCA-B, or POU2AF1) functions as a transcriptional coactivator that specifically associates with either POU2F1/OCT1 or POU2F2/OCT2 transcription factors. It enhances POU2F1/OCT1-mediated promoter activity and, to a lesser extent, that of POU2F2/OCT2 . The protein recognizes the POU domains of these transcription factors and plays an essential role in B-cell response to antigens . BOB1 is required for germinal center formation and regulates IL6 expression in B cells . Its constitutive, B-cell-specific expression pattern makes it a valuable marker for B-cell research and diagnostics .

The strongest expression of BOB1 is typically found in germinal center B cells, mantle-zone B cells, and plasma cells . This differential expression pattern is valuable for identifying and studying various B-cell populations in normal and pathological tissues.

Which tissues and cell types express BOB1?

BOB1 shows consistent expression patterns across specific tissues and cell types:

• Normal tissues: Strong nuclear BOB1 expression is observed in germinal center B cells, with moderate staining in scattered mantle zone B cells and interfollicular T cells .

• B-cell lineage: BOB1 is expressed at various stages of B-cell development, with the highest levels in germinal center-derived cells .

• Lymphomas: Expression patterns vary across different lymphoma types:

  • Nodular lymphocyte predominant Hodgkin lymphoma: LP cells consistently show BOB1 immunopositivity .

  • Classical Hodgkin lymphoma: 94% of cases show moderate to strong nuclear BOB1 positive Hodgkin/Reed-Sternberg cells .

  • Other B-cell lymphomas: Follicular center cell lymphoma and diffuse large B-cell lymphoma show high BOB1 expression, while B-CLL, marginal zone lymphoma, and mantle cell lymphoma typically display weak to moderate immunoreactivity .

What are the recommended applications for BOB1 antibody detection?

BOB1 antibody can be utilized in multiple experimental applications:

• Immunohistochemistry (IHC): Particularly effective for formalin-fixed, paraffin-embedded tissue sections. Recommended concentration ranges from 1-3 μg/ml for optimal staining of germinal center B-cells in human spleen sections .

• Western Blot (WB): Successfully detects an approximately 37kDa band in B-cell line lysates with recommended concentrations of 0.5-1.5 μg/ml .

• Immunocytochemistry (ICC): Effective for staining nuclei in B-cell lines like Raji, with recommended concentrations of 1-3 μg/ml .

• Protein Array (PA): Used for specificity testing against large protein panels .

Epitope retrieval methods significantly impact staining quality; boiling at pH6 for 10-20 minutes followed by 20 minutes of cooling is recommended for IHC applications .

What are the optimal protocols for BOB1 antibody staining in immunohistochemistry?

For optimal BOB1 immunohistochemical staining, follow this methodological approach:

• Sample preparation: Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness) .

• Epitope retrieval: Heat-induced epitope retrieval is recommended. Specifically, boiling at pH6 for 10-20 minutes followed by 20 minutes cooling provides optimal antigen exposure .

• Antibody concentration: Apply BOB1 antibody at 1-2 μg/ml for 30 minutes at room temperature .

• Detection system: DAB staining using HRP polymer provides clear visualization .

• Blocking: For rabbit monoclonal antibodies, 5% NFDM/TBST is recommended as a blocking/dilution buffer .

• Controls: Tonsil and lymph node tissues serve as appropriate positive controls .

Characteristic staining patterns include nuclear and perinuclear positivity in germinal center B-cells, with cytoplasmic staining in some cases .

How should researchers troubleshoot weak or non-specific BOB1 staining?

When encountering staining issues with BOB1 antibody, consider these methodological adjustments:

• Weak or absent staining:

  • Verify antibody concentration (increase from 1 μg/ml to 3 μg/ml if signal is weak) .

  • Extend incubation time from 30 minutes to 60 minutes at room temperature.

  • Optimize epitope retrieval conditions: ensure complete deparaffinization and try longer heat-induced epitope retrieval times .

  • Ensure tissue fixation was adequate (overfixation can mask epitopes).

• Non-specific background staining:

  • Increase blocking duration and concentration (5% NFDM/TBST is recommended) .

  • Reduce primary antibody concentration.

  • Ensure proper washing between steps (minimum 3x5 minutes with TBST).

  • Use isotype-matched control antibodies to confirm specificity.

  • Consider using monospecific antibodies tested against >19,000 full-length human proteins for highest specificity .

• Inconsistent results between experiments:

  • Standardize fixation times and processing parameters.

  • Use automated staining platforms for consistency.

  • Document lot-to-lot variability by maintaining control tissue sections.

What are the differences between mouse and rabbit monoclonal BOB1 antibodies?

Mouse and rabbit monoclonal antibodies against BOB1 have distinct characteristics that impact experimental applications:

When selecting between these antibodies, consider:

• Mouse monoclonals may be preferable for multicolor immunofluorescence when combining with rabbit antibodies against other targets.

• Rabbit monoclonals often demonstrate higher affinity and may require lower working concentrations.

• The specific clone selection should be guided by application requirements and validation data for the intended tissue type .

How does BOB1 expression correlate with EBV status in lymphomas?

BOB1 expression demonstrates a significant correlation with Epstein-Barr virus (EBV) status in lymphomas, particularly in classic Hodgkin lymphoma (CHL):

• Expression correlation: A close association exists between BOB1 immunoreactivity and EBV viral load in CHL cases (p < 0.001) .

• Expression patterns by subtype:

  CHL Subtype	BOB-1 Positive	EBV EBER Positive	EBV LMP-1 Positive
  LRHL	2/2 (100%)	2/2 (100%)	1/2 (50%)
  MC	4/4 (100%)	3/4 (75%)	3/4 (75%)
  NS	9/10 (90%)	6/10 (60%)	6/10 (60%)
  LD	2/2 (100%)	1/2 (50%)	1/2 (50%)
  Total	17/18 (94%)	12/18 (67%)	11/18 (61%)

• Detection methodology: When investigating this correlation, researchers should employ:

  • Nuclear BOB1 immunohistochemistry

  • Nuclear EBER in-situ hybridization

  • Membranous/cytoplasmic/paranuclear LMP-1 immunohistochemistry

This correlation suggests potential functional interactions between EBV infection and BOB1 expression in lymphoma pathogenesis, presenting opportunities for further mechanistic studies in EBV-associated lymphomas.

How can BOB1 antibody be used in differential diagnosis of lymphoma subtypes?

BOB1 antibody serves as a valuable diagnostic tool for differentiating lymphoma subtypes when used in appropriate diagnostic algorithms:

• Hodgkin lymphoma differentiation:

  • Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL): Lymphocyte predominant (LP) cells consistently express BOB1 as they are germinal center-derived .

  • Classical Hodgkin lymphoma (CHL): While 94% of cases show BOB1 expression, the intensity and pattern differ from NLPHL .

  • Primary mediastinal B-cell lymphoma vs. classical Hodgkin's disease: BOB1 expression patterns help distinguish between these entities .

• B-cell lymphoma classification:

  • Germinal center-derived lymphomas (follicular lymphoma, Burkitt lymphoma, some DLBCL): Strong nuclear BOB1 expression .

  • Non-germinal center lymphomas (B-CLL, marginal zone lymphomas, mantle cell lymphomas): Negative or heterogeneous/weaker BOB1 expression .

• Diagnostic panel approach: BOB1 antibody should be incorporated into a comprehensive panel including:

  • Other B-cell markers (CD20, CD79a, PAX5)

  • Germinal center markers (BCL6, CD10)

  • Transcription factors (OCT2, which functions with BOB1)

  • EBV markers (EBER-ISH, LMP-1)

The diagnostic value is maximized when interpreting BOB1 staining patterns alongside morphological features and other immunohistochemical markers, rather than as an isolated finding.

What are the methodological considerations for multiplex immunofluorescence using BOB1 antibody?

When implementing multiplex immunofluorescence (mIF) protocols incorporating BOB1 antibody, researchers should address these methodological considerations:

• Antibody selection and validation:

  • Choose BOB1 antibody clones validated specifically for immunofluorescence applications .

  • Verify absence of cross-reactivity with other targets in the multiplex panel.

  • AE00200 has been successfully used for immunofluorescence with CF488 (green) detection alongside nuclear RedDot counterstaining .

• Panel design and fluorophore selection:

  • Pair BOB1 with complementary B-cell markers (CD20, PAX5) and transcription factors (OCT2).

  • Choose spectrally distinct fluorophores to minimize bleed-through.

  • Consider the nuclear/perinuclear localization of BOB1 when selecting markers with different subcellular localization patterns.

• Sequential staining considerations:

  • Test antibody order effects to determine optimal staining sequence.

  • Incorporate appropriate epitope retrieval between antibody applications if using sequential staining.

  • Validate multiplexed results against single-stain controls.

• Image acquisition and analysis:

  • Utilize confocal microscopy for optimal resolution of nuclear and perinuclear signals .

  • Implement spectral unmixing algorithms for closely overlapping fluorophores.

  • Develop quantitative analysis frameworks to assess co-expression patterns.

• Controls for multiplexed staining:

  • Include single-stained controls for each antibody.

  • Use FMO (fluorescence minus one) controls.

  • Incorporate isotype controls and unstained tissue autofluorescence controls.

How do different BOB1 antibody clones compare in sensitivity and specificity?

When evaluating different BOB1 antibody clones, researchers should consider these comparative performance characteristics:

• Clone-specific properties:

  • AE00200 (Mouse Monoclonal): Demonstrates specificity against >19,000 full-length human proteins, with recommended concentrations of 1-3 μg/ml for IHC and ICC applications .

  • EPR17685 (Rabbit Monoclonal): Effective at 1/10000 dilution for Western blot applications with RAMOS cell lysates .

  • ZM74 (Mouse Monoclonal): Validated as monospecific for diagnostic IHC applications on formalin-fixed, paraffin-embedded tissues .

  • BOB1/2423 (Mouse Monoclonal): Available with various fluorescent labels, including CF® dyes for enhanced brightness and photostability .

  • SP92 (Rabbit Monoclonal): Alternative rabbit monoclonal with similar applications to EPR17685 .

• Cross-reactivity considerations:

  • Most clones are primarily validated for human samples .

  • Cross-reactivity with other species should be experimentally verified.

  • Protein BLAST searches against the target species can predict potential cross-reactivity issues .

• Selection criteria for research applications:

  • Application requirements (IHC, WB, ICC)

  • Host species (to avoid cross-reactivity with secondary antibodies)

  • Isotype (important for multiplexed applications)

  • Validated subcellular localization patterns

  • Known performance in specific tissue types

Researchers should conduct side-by-side comparisons when transitioning between clones and maintain detailed records of performance differences to ensure experimental reproducibility.

What are the considerations for combined BOB1 and OCT2 immunostaining in lymphoma research?

For effective combined BOB1 and OCT2 immunostaining in lymphoma research, consider these methodological approaches:

• Biological relevance: BOB1 and OCT2 form a functional complex in B-cells, with BOB1 serving as a coactivator for OCT2-mediated transcription . Their combined expression pattern provides more comprehensive insights into B-cell functionality than either marker alone.

• Sequential vs. simultaneous staining:

  • Sequential approach: Apply antibodies in separate staining rounds with an intermediary epitope retrieval step. This minimizes cross-reactivity but may impact tissue integrity.

  • Simultaneous approach: Requires careful selection of primary antibodies from different host species and compatible detection systems.

• Interpretation guidelines:

  • Germinal center-derived lymphomas typically show strong nuclear expression of both BOB1 and OCT2 .

  • In classical Hodgkin lymphoma, Hodgkin/Reed-Sternberg cells may express only one or neither protein .

  • Discordant expression (OCT2+/BOB1- or OCT2-/BOB1+) may indicate specific pathological states.

• Technical recommendations:

  • Use spectral unmixing for fluorescent co-localization studies.

  • For chromogenic double staining, select contrasting chromogens (e.g., DAB for one marker and Fast Red for the other).

  • Implement digital image analysis to quantify co-expression patterns.

• Validation approaches:

  • Compare immunostaining results with mRNA expression data.

  • Correlate with functional assays measuring OCT2/BOB1-dependent transcriptional activity.

  • Include known positive and negative control tissues in each experiment.

How can researchers quantify and interpret variable BOB1 expression in different B-cell populations?

To accurately quantify and interpret variable BOB1 expression across B-cell populations, researchers should implement these methodological approaches:

• Standardized scoring systems:

  • Intensity scoring: Develop a 4-tier system (0=negative, 1=weak, 2=moderate, 3=strong) for nuclear staining intensity.

  • Percentage scoring: Record the percentage of positive cells within each population (25-100% positivity has been observed in Hodgkin lymphoma) .

  • H-score method: Calculate H-score = Σ(intensity × percentage) for quantitative comparison.

• Digital pathology approaches:

  • Implement whole slide imaging with machine learning algorithms for consistent scoring.

  • Use multiplex image analysis to correlate BOB1 expression with other B-cell markers.

  • Apply spatial analysis to evaluate expression patterns in different microenvironmental contexts.

• Alternative quantification methods:

  • Flow cytometry: For fresh or frozen samples, quantify BOB1 expression at the single-cell level.

  • Western blotting: For bulk tissue analysis with densitometric quantification.

  • qRT-PCR: To correlate protein expression with mRNA levels.

• Interpretation frameworks:

  • Compare expression patterns against known reference tissues (tonsil, lymph node) .

  • Consider developmental stage-specific expression patterns in normal B-cell maturation.

  • Account for microenvironmental influences on expression in different anatomical compartments.

• Biological correlates:

  • Correlate BOB1 expression levels with B-cell activation status.

  • Assess relationship to immunoglobulin production capabilities.

  • Evaluate association with proliferation markers and cell cycle status.

This comprehensive approach enables meaningful comparison of BOB1 expression across different studies and pathological conditions.

What emerging roles might BOB1 antibodies play in understanding B-cell lymphomagenesis?

BOB1 antibodies are positioned to contribute significantly to our understanding of B-cell lymphomagenesis through several emerging research directions:

• Transcriptional network mapping: As a transcriptional coactivator, BOB1's interactions with OCT transcription factors regulate key B-cell genes. Mapping these networks through ChIP-seq and other genomic approaches will reveal how their dysregulation contributes to lymphomagenesis .

• Microenvironmental interactions: The differential expression of BOB1 in various lymphoma subtypes suggests its potential role in tumor-microenvironment interactions. Future studies could explore how BOB1 expression affects interactions with T-cells and other stromal components .

• EBV-mediated lymphomagenesis: The strong correlation between BOB1 expression and EBV viral load (p < 0.001) points to potential mechanistic interactions that could be further elucidated through functional studies .

• Therapeutic target potential: Understanding BOB1's role in maintaining B-cell lymphoma phenotypes may identify novel therapeutic approaches targeting this transcriptional coactivator or its downstream pathways.

• Biomarker development: Refined analysis of BOB1 expression patterns, particularly in combination with other B-cell markers, may lead to more precise prognostic and predictive biomarkers for lymphoma management.

These directions would benefit from technological advances in single-cell analysis, spatial transcriptomics, and computational modeling of transcriptional networks to fully uncover BOB1's role in normal and malignant B-cell biology.

How might future technological advances enhance BOB1 antibody applications in research and diagnostics?

Future technological developments are poised to expand and enhance BOB1 antibody applications in both research and diagnostics:

• Advanced multiplex immunohistochemistry platforms:

  • Highly multiplexed tissue imaging (40+ markers) will enable comprehensive characterization of BOB1 expression in complex B-cell microenvironments.

  • Cyclic immunofluorescence and mass cytometry imaging will allow more comprehensive phenotyping of BOB1-expressing cells.

• Digital pathology integration:

  • AI-based image analysis will standardize BOB1 expression quantification.

  • Machine learning algorithms will identify subtle expression patterns that correlate with clinical outcomes.

  • Cloud-based platforms will facilitate multi-institutional studies of BOB1 expression across large cohorts.

• Single-cell technologies:

  • Integration of BOB1 immunophenotyping with single-cell transcriptomics.

  • Spatial transcriptomics to map BOB1 expression in the context of tissue architecture.

  • Computational approaches linking BOB1 protein expression to gene regulatory networks.

• Novel antibody formats:

  • Development of recombinant antibody fragments with enhanced tissue penetration.

  • Site-specific conjugation strategies for improved fluorophore or nanoparticle labeling.

  • Bispecific antibodies targeting BOB1 and other B-cell markers for enhanced specificity.

• Point-of-care diagnostics:

  • Rapid immunoassays incorporating BOB1 antibodies for expedited lymphoma classification.

  • Microfluidic devices for automated staining and quantification in resource-limited settings.

  • Integration with liquid biopsy approaches for non-invasive monitoring.