TNFSF13 Antibody

What is TNFSF13 and why is it important in immunological research?

TNFSF13, also known as APRIL (a proliferation-inducing ligand), is a member of the TNF ligand superfamily. It functions as a cytokine that binds to two receptors: TNFRSF13B/TACI and TNFRSF17/BCMA . TNFSF13 plays crucial roles in:

• Lymphocyte maturation and physiological activities

• Regulation of tumor cell growth and progression

• Protection from apoptosis in normal and transformed cells

• Immunosuppression via diverse immunoregulatory pathways

• Monocyte/macrophage-mediated immunological processes

In research contexts, TNFSF13 has emerged as an important molecule due to its involvement in various pathological activities, including neoplasia and inflammatory conditions . Its expression has been explored in multiple cancer types, including breast cancer, multiple myeloma, and gliomas, making it a significant target for immunotherapy research .

How should researchers select the appropriate TNFSF13 antibody for their specific experimental applications?

Selecting the appropriate TNFSF13 antibody requires careful consideration of several factors:

By Application:

• Western Blotting: Choose antibodies validated for WB with demonstrated specificity, such as rabbit polyclonal antibodies that recognize specific epitopes within the TNFSF13 protein .

• Immunohistochemistry: For IHC-P applications, antibodies like rabbit polyclonal anti-TNFSF13 (1:500 dilution) have been successfully used in published research .

• Flow Cytometry: For intracellular staining, use antibodies validated for FACS that may require specific fixation and permeabilization protocols .

• ELISA: Consider antibody pairs specifically designed for sandwich ELISA development, such as Mouse Anti-Human APRIL/TNFSF13 Monoclonal Antibody (capture) paired with Goat Anti-Human APRIL/TNFSF13 Polyclonal Antibody (detection) .

By Species Reactivity:

Select antibodies based on your target species. Available TNFSF13 antibodies react with various species:

By Clonality:

Consider whether a monoclonal or polyclonal antibody is more suitable for your specific application .

What are the optimal conditions for using TNFSF13 antibodies in flow cytometry experiments?

For successful flow cytometry experiments using TNFSF13 antibodies, researchers should consider the following protocol guidelines:

Sample Preparation:

• For cellular samples such as U937 human lymphoma cell lines, proper fixation is critical before antibody staining .

• Fix cells with Flow Cytometry Fixation Buffer to maintain cellular integrity.

• For intracellular staining, permeabilize cells with Flow Cytometry Permeabilization/Wash Buffer I .

Staining Protocol:

• Block non-specific binding sites with appropriate blocking buffer.

• For primary antibody incubation, use Goat Anti-Human APRIL/TNFSF13 Antigen Affinity-purified Polyclonal Antibody at a dilution of 1:100 to 1:200 .

• Incubate cells with the primary antibody for 30-60 minutes at room temperature or overnight at 4°C.

• Wash cells thoroughly (3 times) to remove unbound antibody.

• Incubate with appropriate fluorescently-labeled secondary antibody (e.g., anti-Goat IgG PE-conjugated Secondary Antibody).

• Perform final washes before analysis.

Controls and Validation:

Always include appropriate controls:

• Isotype control (e.g., AB-108-C as shown in published protocols)

• Unstained cellular samples

• Single-color controls for compensation if using multiple fluorophores

The detection of TNFSF13 in U937 human cell lines has been successfully demonstrated using this approach, with clear differentiation between TNFSF13-positive cells and control antibody staining .

What are the most effective protocols for detecting TNFSF13 expression in tissue samples using immunohistochemistry?

For optimal immunohistochemical detection of TNFSF13 in tissue samples:

Tissue Preparation:

• Fix tissue samples in formalin and embed in paraffin.

• Section tissues at 4-6 μm thickness and mount on positively charged slides.

• Deparaffinize sections using xylene and rehydrate through graded alcohols.

Antigen Retrieval:

Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes is often effective for TNFSF13 detection.

Staining Protocol:

• Block endogenous peroxidase activity with 3% hydrogen peroxide.

• Block non-specific binding with 5% BSA as demonstrated in published protocols .

• Incubate with primary antibody (rabbit polyclonal anti-TNFSF13 antibody at 1:500 dilution) at 4°C overnight .

• Apply appropriate detection system (e.g., HRP-conjugated secondary antibody).

• Develop with DAB or other suitable chromogen.

• Counterstain, dehydrate, clear, and mount.

Validation and Controls:

• Include positive control tissues with known TNFSF13 expression.

• Include negative controls by omitting primary antibody.

• Consider using competing peptides to verify specificity, as demonstrated in published immunofluorescence experiments .

This approach has been validated in human brain cortex tissue using antibodies at 10 μg/ml concentration with successful visualization of TNFSF13 expression patterns .

How does TNFSF13 expression correlate with immune cell infiltration in tumor microenvironments?

Research has revealed significant correlations between TNFSF13 expression and immune cell infiltration in tumor microenvironments, particularly in gliomas:

Key Correlations:

• TNFSF13 expression positively correlates with:

  • Immune score, stromal score, and ESTIMATE score in pan-glioma and GBM groups

  • Infiltration of various immune cells including:

    • NK cells

    • Monocytes

    • Macrophages (particularly M2 phenotype)

    • CD8+ T effector memory cells (TEM)

    • CD4+ TEM

    • Myeloid-derived suppressor cells (MDSC)

    • Regulatory T cells (Treg)

    • Neutrophils

    • Fibroblasts

• TNFSF13 expression negatively correlates with:

  • Type 2 T helper cells

  • Memory B cells

Immune Checkpoint Associations:

TNFSF13 shows synergistic relationships with multiple immune checkpoint molecules across various cancer types:

• PDCD1LG2

• HAVCR2

• PDCD1

• CD80

• CTLA4

• CD274

• IDO1

• CD276

Immunogram Analysis:

High TNFSF13 expression correlates with elevated scores in multiple immunogram terms:

• Glycolysis

• Innate immunity

• Priming activation

• T cells

• Interferon γ response

• Inhibitory molecules

• Inhibitory cells (Tregs and MDSCs)

• Recognition of tumor cells

These findings suggest TNFSF13 may function as an onco-inflammatory marker and could serve as a potential biomarker for immunotherapy response in cancer treatment.

What methodological approaches should be used to validate TNFSF13 antibody specificity?

Rigorous validation of TNFSF13 antibody specificity is crucial for reliable research outcomes. Recommended methodological approaches include:

Western Blot Validation:

• Use positive control samples with known TNFSF13 expression.

• Include negative controls (tissues/cells with low or no TNFSF13 expression).

• Verify band size corresponds to expected molecular weight of TNFSF13 (17 kDa for secreted form, 32 kDa for transmembrane form) .

• Perform peptide competition assays:

  • Pre-incubate antibody with synthesized TNFSF13 peptide

  • Compare results with non-blocked antibody staining

Cell and Tissue Panel Screening:

Test antibody against multiple cell lines and tissue types with varying TNFSF13 expression levels to demonstrate consistent and specific detection patterns.

Knockout/Knockdown Validation:

• Compare antibody reactivity in wild-type versus TNFSF13 knockout or knockdown models.

• As demonstrated in TNFSF13 variant studies, reduced TNFSF13 expression should correspond with reduced antibody signal .

Cross-Reactivity Assessment:

Test for cross-reactivity with closely related proteins, particularly BAFF, which shows sequence homology with TNFSF13/APRIL .

Multiple Detection Methods:

Confirm specificity using complementary techniques:

• If an antibody shows specific staining in Western blot, confirm with IHC or IF

• Compare results from different antibody clones targeting different epitopes of TNFSF13

A thorough validation strategy employing multiple approaches provides the strongest evidence for antibody specificity and research reliability.

How can researchers effectively use TNFSF13 antibodies in single-cell RNA sequencing and imaging mass cytometry studies?

Advanced research incorporating TNFSF13 antibodies in cutting-edge single-cell technologies requires specific methodological considerations:

Single-Cell RNA Sequencing (scRNA-seq):

• Cell Preparation and Sorting:

  • Isolate cells from fresh tissue biopsies or colonoid cultures

  • Consider using TNFSF13 antibodies for pre-enrichment of specific cell populations

• Data Analysis Strategies:

  • Identify cell clusters using established markers

  • Analyze TNFSF13 expression across different cell populations (as demonstrated in published research showing TNFSF13 upregulation in M2 macrophages, T cells, and cancer cells)

  • Perform pseudotime trajectory analysis to reveal developmental relationships between cell states

  • Correlate TNFSF13 expression with other genes of interest

• Validation Approaches:

  • Confirm scRNA-seq findings with protein-level detection using flow cytometry or imaging techniques

  • Compare expression patterns between patient samples and appropriate controls

Imaging Mass Cytometry (IMC):

• Panel Design:

  • Include TNFSF13 antibody alongside markers for:

    • T cells (CD3, CD4, CD8)

    • Regulatory T cells (FOXP3)

    • B cells (CD20)

    • Plasma cells

    • Myeloid cells

    • Dendritic cells

    • Macrophages

    • Proliferation markers (Ki67)

• Spatial Analysis:

  • Retain X and Y coordinates of each cell to assess spatial relationships

  • Analyze immune cell composition with spatial resolution (as demonstrated in studies showing CD20+ B cells near epithelial crypts in TNFSF13 variant tissue)

  • Quantify distances between different cell types to identify potential interactions

• Multiplexed Analysis:

  • Combine TNFSF13 detection with markers for proliferation to assess effects on cell growth

  • Analyze co-expression patterns to identify cellular subpopulations

These advanced methodologies have successfully revealed novel insights into TNFSF13 function, including its role in B cell recruitment and differentiation in mucosal tissues.

What are the critical differences between polyclonal and monoclonal TNFSF13 antibodies for specific research applications?

Understanding the distinct properties of polyclonal versus monoclonal TNFSF13 antibodies is crucial for selecting the appropriate reagent for specific applications:

Polyclonal TNFSF13 Antibodies:

Characteristics:

• Recognize multiple epitopes on the TNFSF13 protein

• Often generated in goat, rabbit, or other host species

• Examples: Goat Anti-Human APRIL/TNFSF13 Antigen Affinity-purified Polyclonal Antibody (AF884)

Optimal Applications:

• Western blotting: Superior sensitivity for detecting denatured proteins

• Immunoprecipitation: Better capturing capability due to multiple epitope recognition

• Detection antibody in ELISA pairs: Enhanced signal amplification

• Immunohistochemistry: Often provides stronger signal in tissue sections

Limitations:

• Batch-to-batch variability

• Potential for higher background in some applications

• May show cross-reactivity with closely related proteins

Monoclonal TNFSF13 Antibodies:

Characteristics:

• Recognize a single epitope on TNFSF13

• Often generated in mouse

• Examples: Mouse Anti-Human APRIL/TNFSF13 Monoclonal Antibody (Clone 540218, MAB8844)

Optimal Applications:

• Capture antibody in ELISA pairs: Provides consistent specificity

• Flow cytometry: Lower background and reliable performance

• Applications requiring high reproducibility

• Therapeutic applications (e.g., sibeprenlimab, a humanized IgG2SA monoclonal antibody)

Limitations:

• May have lower sensitivity for certain applications

• Potential for epitope masking in native conformations

• May be less effective if target epitope is altered by fixation or processing

Application-Specific Recommendations:

For critical applications, researchers should compare the performance of both antibody types to determine which provides optimal results for their specific experimental system.

How does TNFSF13 antibody performance vary across different sample types and preparation methods?

TNFSF13 antibody performance can vary significantly depending on sample types and preparation methods, which is critical knowledge for experimental design:

Cell Lines vs. Primary Tissues:

Cell Lines:

• Generally provide more consistent TNFSF13 detection

• U937 human lymphoma cell line shows reliable TNFSF13 expression detectable by flow cytometry

• HeLa cells display TNFSF13 expression detectable by immunofluorescence

• Require fixation and permeabilization for intracellular staining

Primary Tissues:

• Show variable TNFSF13 expression based on tissue type and disease state

• Human brain cortex tissue requires specific IHC conditions (10 μg/ml antibody concentration)

• Colonic epithelial cells show distinct TNFSF13 expression patterns that can be altered in variant tissues

• Often require more rigorous antigen retrieval methods

Fresh vs. Fixed Samples:

Fresh/Frozen Samples:

• Often yield stronger signals with less epitope masking

• Preferred for certain applications (e.g., immunoprecipitation)

• May require different antibody concentrations than fixed samples

Fixed Samples:

• Formalin fixation can mask TNFSF13 epitopes, requiring appropriate antigen retrieval

• Paraffin embedding preserves tissue architecture but may reduce antibody accessibility

• Successful detection demonstrated in fixed human brain cortex tissue

3D Cultures and Organoids:

Studies using colonoid cultures have demonstrated:

• Successful TNFSF13 antibody staining in 3D culture systems

• TNFSF13 variant colonoids show altered expression of markers like ALDOB and FABP2

• Requires optimization of penetration and washing steps

Sample Preparation Recommendations:

Optimization for each specific application and sample type is essential, with titration experiments recommended to determine ideal antibody concentrations.

What methodological approaches should be used to investigate TNFSF13 function in tumor immunology research?

Investigating TNFSF13 function in tumor immunology requires multifaceted methodological approaches:

Expression Analysis in Tumor Microenvironment:

Single-Cell Resolution Techniques:

• Single-cell RNA sequencing to identify TNFSF13-expressing cells within tumors

• Imaging mass cytometry to spatially resolve TNFSF13+ cells relative to other immune populations

• Flow cytometry with intracellular staining for TNFSF13 in different immune subsets

Spatial Analysis:

• Multiplex immunofluorescence to visualize TNFSF13 expression relative to:

  • Tumor cells

  • Immune checkpoints (PDCD1, CTLA4, CD274)

  • Immune cell infiltrates (macrophages, T cells, B cells)

Functional Assessment of TNFSF13 in Immune Regulation:

In Vitro Approaches:

• Co-culture systems with:

  • TNFSF13-expressing cells and immune effector cells

  • Blocking antibodies to assess TNFSF13-dependent effects

  • Recombinant TNFSF13 protein to examine dose-dependent responses

Receptor Engagement Analysis:

• Evaluate TNFSF13 binding to BCMA and TACI receptors

• Assess downstream signaling pathways

• Quantify effects on immune cell proliferation, survival, and function

In Vivo Models and Clinical Correlations:

Genetic Approaches:

• TNFSF13 knockout or variant models to assess impacts on tumor growth and immune infiltration

• CRISPR-engineered tumor cells with altered TNFSF13 expression

Therapeutic Targeting:

• TNFSF13-blocking antibodies (e.g., sibeprenlimab) in preclinical models

• Combination approaches with immune checkpoint inhibitors

Clinical Correlation Studies:

• Analysis of TNFSF13 expression in relation to:

  • Patient survival (as demonstrated in glioma studies)

  • Immune infiltration patterns

  • Response to immunotherapy

  • Association with ESTIMATE scores, MMR, MSI, and TMB

Key Research Areas Based on Recent Findings:

TNFSF13 as an Immunosuppressive Regulator:

• Studies show TNFSF13 is associated with immunogram terms including:

  • Inhibitory molecules

  • Inhibitory cells (Tregs and MDSCs)

  • T cell immunity modulation

TNFSF13 in B Cell Function:

• Research demonstrates TNFSF13 affects:

  • B cell accumulation near epithelial crypts

  • Plasma cell differentiation

  • IgA production

By employing these comprehensive methodological approaches, researchers can gain deeper insights into TNFSF13's role in tumor immunology and potentially develop targeted therapeutic strategies.

How can researchers troubleshoot common issues when using TNFSF13 antibodies in experimental procedures?

Effectively troubleshooting issues with TNFSF13 antibodies requires systematic approaches to identify and resolve common problems:

Weak or No Signal:

Potential Causes and Solutions:

• Insufficient Antigen Expression

  • Verify TNFSF13 expression in your sample using positive controls (U937 cells show reliable expression)

  • Consider using samples with known higher expression (e.g., certain immune cells or tumor tissues)

• Epitope Masking

  • For fixed tissues: Optimize antigen retrieval methods (citrate or EDTA buffer)

  • For Western blot: Ensure complete protein denaturation

  • Try antibodies targeting different TNFSF13 epitopes

• Antibody Concentration

  • Titrate antibody concentration (successful IHC has been reported at 1:500 dilution or 10 μg/ml)

  • For Western blot: Optimal concentration reported at 0.5 μg/mL

• Detection System

  • Ensure secondary antibody matches host species of primary antibody

  • Consider signal amplification methods (e.g., biotin-streptavidin systems)

  • For ELISA: Verify optimal antibody pair combinations

High Background:

Potential Causes and Solutions:

• Non-specific Binding

  • Optimize blocking (5% BSA has been successfully used)

  • Increase washing steps duration/frequency

  • Reduce antibody concentration

  • Include appropriate negative controls

• Cross-Reactivity

  • Test for cross-reactivity with related proteins (especially BAFF)

  • Use monoclonal antibodies for higher specificity

  • Perform peptide competition assays to confirm specificity

• Sample-Specific Issues

  • For tissues with high endogenous peroxidase: Optimize quenching step

  • For tissues with endogenous biotin: Use biotin-free detection systems

Inconsistent Results:

Potential Causes and Solutions:

• Antibody Stability

  • Follow manufacturer's storage recommendations

  • Avoid repeated freeze-thaw cycles

  • Consider aliquoting antibodies

• Sample Variability

  • Standardize fixation and processing protocols

  • Use consistent cell culture conditions

  • Account for potential heterogeneity in TNFSF13 expression within tissues

• Protocol Consistency

  • Document detailed protocols

  • Control incubation times and temperatures precisely

  • Use automated systems where possible

Application-Specific Troubleshooting:

Methodical troubleshooting with appropriate controls is essential for resolving issues with TNFSF13 antibody applications.

What are the emerging research applications of TNFSF13 antibodies in studying immunotherapeutic approaches?

TNFSF13 antibodies are increasingly utilized in cutting-edge immunotherapy research, revealing new applications and therapeutic possibilities:

Targeting TNFSF13 in Combination Immunotherapy:

Recent research has revealed that TNFSF13 expression correlates with multiple immune checkpoint molecules, suggesting potential for combination therapies:

• Synergistic Mechanisms: TNFSF13 shows close association with PDCD1LG2, HAVCR2, PDCD1, CD80, CTLA4, CD274, IDO1, and CD276 across multiple cancer types

• Therapeutic Implications: This suggests TNFSF13-targeting antibodies like sibeprenlimab could enhance efficacy of existing checkpoint inhibitors

• Research Applications: TNFSF13 antibodies can be used to study mechanisms of synergy between different immunomodulatory pathways

Biomarker Development for Immunotherapy Response:

TNFSF13 expression correlates with established biomarkers of immunotherapy efficacy:

• Correlation Studies: Research demonstrates relationships between TNFSF13 and:

  • Mismatch repair (MMR)

  • Microsatellite instability (MSI)

  • Tumor mutational burden (TMB)

• Methodological Approaches: Antibody-based detection of TNFSF13 in patient samples can:

  • Help stratify patients for immunotherapy

  • Predict potential responders

  • Monitor treatment efficacy

Modulation of Tumor Microenvironment:

TNFSF13 antibodies enable research into tumor microenvironment modulation:

• Macrophage Polarization: Studies show TNFSF13 affects M2 macrophage development, a target for reprogramming the immunosuppressive microenvironment

• B Cell Function: Research with TNFSF13 antibodies reveals roles in:

  • B cell recruitment to tumor sites

  • Plasma cell differentiation

  • Antibody production within the tumor microenvironment

• T Cell Activity: TNFSF13 affects regulatory T cell differentiation and T helper 1 cell function

Novel Therapeutic Antibody Development:

Research-grade antibodies inform development of therapeutic candidates:

• Humanized Antibodies: Research with sibeprenlimab (humanized IgG2SA anti-TNFSF13) demonstrates potential for therapeutic development

• Epitope Mapping: Antibodies targeting different TNFSF13 domains help identify optimal binding sites for therapeutic intervention

• Functional Blocking: Research using antibodies that block TNFSF13-receptor interactions inform therapeutic mechanism design

Clinical Translation Research:

TNFSF13 antibody research facilitates clinical translation:

• Immunohistochemical Analysis: Patient biopsies analyzed with TNFSF13 antibodies reveal:

  • Expression patterns across cancer types

  • Associations with prognosis

  • Correlation with treatment response

• Liquid Biopsy Development: Research exploring detection of soluble TNFSF13 in patient serum as less invasive biomarker

• Companion Diagnostics: Development of antibody-based assays to identify patients likely to benefit from TNFSF13-targeted therapies