C4BPA Antibody

What is C4BPA and what roles does it play in biological systems?

C4BPA is a 67 kDa protein (observed at ~70 kDa in Western blots) that forms part of the C4b-binding protein complex. It primarily functions as a regulator of the classical complement pathway by:

• Binding as a cofactor to C3b/C4b inactivator (C3bINA), which hydrolyzes complement fragment C4b

• Accelerating degradation of the C4bC2a complex (C3 convertase) by dissociating C2a

• Interacting with anticoagulant protein S and serum amyloid P component

Beyond its canonical role in complement regulation, C4BPA has emerged as an important immune modulator through its interaction with CD40. This interaction promotes T cell proliferation and induces anti-tumor immune responses . Additionally, C4BPA has been identified as a potential biomarker in multiple cancer types, including breast cancer, pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer .

Recent research has also revealed that C4BPA can be expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptotic pathways . This dual localization (extracellular and intracellular) makes C4BPA a particularly interesting target for cancer research.

What types of C4BPA antibodies are available for research applications?

Researchers have access to several types of C4BPA antibodies, each with specific applications and characteristics:

When selecting a C4BPA antibody, researchers should consider:

• The specific application (Western blot, IHC, flow cytometry, etc.)

• The epitope location (especially important when studying specific domains or interactions)

• The species of interest (most commercial antibodies target human C4BPA)

• The format required (unconjugated vs. conjugated)

• Validation data for your specific application and sample type

How is C4BPA expression altered in cancer compared to normal tissues?

C4BPA expression shows distinct patterns across different cancer types, with important implications for diagnosis and prognosis:

Breast Cancer:

Pancreatic Ductal Adenocarcinoma (PDAC):

• C4BPA has been identified as a serum biomarker for early detection of PDAC

• Stromal C4BPA strongly correlates with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001)

• Recombinant human C4BPA stimulation increases CD4+ and CD8+ T cell numbers in peripheral blood mononuclear cells (PBMCs)

Colorectal Cancer:

• Specific mutations in C4BPA (e.g., 182C>T and 563G>A) affect protein expression levels

• The 182C>T mutation is associated with marked induction of C4BPA protein levels following oxaliplatin treatment

• The 563G>A mutation leads to high baseline C4BPA expression with minimal further increase following treatment

Non-Small Cell Lung Cancer:

• C4BP has been linked to aggressive tumor characteristics

• Associated with poor prognosis in individuals with stage IIb and IIIa disease

These cancer-specific expression patterns make C4BPA an attractive target for both biomarker development and therapeutic interventions.

How can I optimize immunohistochemical detection of C4BPA in tissue samples?

Successful immunohistochemical detection of C4BPA requires careful optimization of several parameters:

Antigen Retrieval Methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative option: Citrate buffer pH 6.0

Antibody Selection and Dilution:

• For polyclonal antibodies: Start with 1:20-1:200 dilution range

• For monoclonal antibodies: Follow manufacturer's recommendations, typically 1:100-1:1000

• Perform titration experiments to determine optimal concentration for your specific tissue

Detection Systems:

• HRP-conjugated secondary antibodies

• VECTASTAIN® Elite® ABC Kit or similar visualization systems

• Develop with 0.01% 3,3-diaminobenzidine

• Counterstain with hematoxylin and eosin

Positive Controls:

• Human liver tissue serves as an excellent positive control

• In pancreatic tissue, islet cells can function as internal positive controls

Scoring System for C4BPA Expression in Cancer Studies:

• Low expression: staining intensity of stroma around cancer cells less than that of islet cells

• High expression: staining intensity of stroma around cancer cells greater than or equal to that of islet cells

For cancer studies, consider dual staining with immune cell markers (CD8, CD4, etc.) to analyze the relationship between C4BPA expression and immune infiltration. This approach has revealed important correlations, particularly in PDAC where stromal C4BPA strongly correlates with CD8+ tumor-infiltrating lymphocytes .

What are the best methods for studying C4BPA interactions with immune components?

C4BPA has significant interactions with various immune components, most notably through the C4BPA-CD40 axis. Here are methodological approaches to investigate these interactions:

Flow Cytometry Analysis:

• Design panels that include C4BPA along with immune cell markers (CD4, CD8, B cell markers)

• Analyze how recombinant human C4BPA (rhC4BPA) affects immune cell populations in PBMCs

• Compare results with mC4BPA peptide (30 amino acids from C-terminus that binds CD40)

Co-immunoprecipitation Studies:

• Use antibodies suitable for IP (e.g., Rabbit Recombinant Monoclonal at 1/60 dilution)

• Probe for known interaction partners: CD40, NFKBIA, RELA, NFKB1, C4BPB, and CFI

• Include appropriate controls (isotype control, reverse IP)

Functional Assays:

• T Cell Proliferation: Measure CD4+ and CD8+ T cell proliferation following rhC4BPA stimulation

• Apoptosis Assays: Assess how combined treatment with anti-cancer drugs (e.g., gemcitabine) and rhC4BPA affects cancer cell apoptosis

• In vivo studies: Use mouse models to evaluate how mC4BPA peptide affects CD8+ tumor-infiltrating lymphocytes

Correlation Analysis with Immune Infiltration:

Based on comprehensive analyses, C4BPA expression correlates significantly with multiple immune cell types:

These correlations can be analyzed using the single-sample Gene Set Enrichment Analysis (ssGSEA) method with the gene set variation analysis (GSVA) package in R for 24 different types of immune cells in tumor samples .

How can I distinguish between intracellular and extracellular C4BPA in research studies?

C4BPA exhibits both extracellular and intracellular functions, requiring different experimental approaches to distinguish between these pools:

For Extracellular C4BPA Detection:

• Serum/Plasma Analysis:

  • ELISA kits specifically designed for C4BPA (detection range: 0.469 - 30 ng/ml)

  • Western blot analysis of plasma/serum samples (observed at ~70 kDa)

  • Consider native complex structure (570 kDa complex of 7 alpha chains and 1 beta chain)

• Cell Surface Expression:

  • Flow cytometry with non-permeabilizing protocols

  • Cell surface biotinylation to selectively label extracellular proteins

For Intracellular C4BPA Detection:

• Cell Preparation:

  • Use permeabilization buffers compatible with intracellular staining

  • For flow cytometry, follow specific intracellular staining protocols ("Flow Cyt (Intra)")

  • For Western blot, ensure complete cell lysis to release intracellular C4BPA

• Subcellular Localization:

  • Perform cellular fractionation to separate nuclear, cytoplasmic, and membrane fractions

  • Western blot analysis of each fraction with C4BPA antibodies

  • Immunofluorescence microscopy with co-staining for cellular compartment markers

Experimental Design Considerations:

• Mutation Studies:

  • Specific mutations (e.g., 182C>T, 563G>A) can affect intracellular C4BPA levels

  • The 182C>T mutation shows marked induction of C4BPA following oxaliplatin treatment

  • The 563G>A mutation exhibits high baseline expression with minimal further increase following treatment

• Stress Response Analysis:

  • Treatment with chemotherapeutic agents (e.g., oxaliplatin) can alter intracellular C4BPA levels

  • Design experiments with appropriate time points to capture dynamic changes in expression

• Interaction Studies:

  • Intracellular C4BPA interacts with RelA (NF-κB family member)

  • Extracellular C4BPA interacts with CD40, protein S, and complement components

  • Design co-immunoprecipitation studies targeting specific interaction partners to distinguish location

How can C4BPA antibodies be used to investigate its role in NF-κB signaling?

Recent research has revealed that intracellular C4BPA interacts with the NF-κB family member RelA and regulates apoptosis. Here are methodological approaches to investigate this relationship:

Protein-Protein Interaction Studies:

• Co-immunoprecipitation:

  • Immunoprecipitate C4BPA and probe for RelA

  • Perform reverse co-IP (immunoprecipitate RelA and probe for C4BPA)

  • Include appropriate controls (isotype antibodies, input samples)

• Proximity Ligation Assay (PLA):

  • Use antibodies against C4BPA and RelA from different species

  • Apply species-specific secondary antibodies with oligonucleotide probes

  • Visualize interaction as fluorescent spots if proteins are in close proximity

Cellular Localization Studies:

• Nuclear-Cytoplasmic Fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Use C4BPA antibodies to detect its presence in different cellular compartments

  • Compare with RelA localization under various conditions

• Immunofluorescence Microscopy:

  • Perform dual staining for C4BPA and RelA

  • Analyze co-localization patterns in different cell compartments

  • Monitor changes in response to NF-κB activating stimuli

Functional Analysis:

• NF-κB Reporter Assays:

  • Measure NF-κB activity using luciferase reporter systems

  • Manipulate C4BPA levels (overexpression, knockdown) and assess effects on NF-κB signaling

  • Use C4BPA antibodies to confirm expression levels

• Apoptosis Assays:

  • As described in the research, C4BPA regulates NF-κB-dependent apoptosis

  • Use flow cytometry with Annexin V/PI staining to measure apoptosis rates

  • Compare wild-type cells with those expressing C4BPA mutations (e.g., 182C>T, 563G>A)

CRISPR-Modified Cell Lines:

• Research has developed CRISPR knock-in cell lines with specific C4BPA mutations:

  • HCT 116 C4BPA 182C>T cells (increased expression following oxaliplatin)

  • HCT 116 C4BPA 563G>A cells (high baseline expression)

• These models allow direct comparison of how different mutations affect NF-κB signaling

What considerations are important when validating C4BPA antibody specificity?

Thorough validation of C4BPA antibody specificity is essential for reliable research outcomes. Here are comprehensive recommendations:

Multiple Validation Approaches:

• Western Blot Validation:

  • Confirm single band at expected molecular weight (~70 kDa)

  • Test in appropriate positive control samples (human plasma/serum)

  • Include C4BPA knockdown/knockout controls

• Genetic Modification Controls:

  • siRNA knockdown of C4BPA (as used in experiments described in search results)

  • CRISPR/Cas9 knockout cell lines

  • CRISPR knock-in cell lines with specific mutations (e.g., 182C>T, 563G>A)

• Preabsorption Tests:

  • Pre-incubate antibody with excess recombinant C4BPA

  • Signal should be dramatically reduced or eliminated in preabsorbed samples

Tissue and Sample Selection:

• Positive Controls:

  • Human plasma/serum (contains high levels of C4BPA)

  • Human liver tissue

  • Islet cells in pancreatic tissue (internal positive control)

• Antibody Controls:

  • Isotype control antibodies matching the C4BPA antibody class

  • Secondary antibody-only controls to check for non-specific binding

  • Multiple antibodies targeting different epitopes of C4BPA

Special Considerations for C4BPA:

• Complex Formation:

  • C4BPA forms complexes with other proteins (C4BPB, protein S)

  • These interactions may affect antibody binding

  • Consider native vs. denatured conditions in different applications

• Mutation Effects:

  • Mutations in C4BPA (as identified in colorectal cancer) may alter epitopes

  • Different mutations (e.g., 182C>T vs. 563G>A) have different effects on protein levels

  • Consider the location of mutations relative to antibody epitopes

• Expression Regulation:

  • Expression is regulated in a stress- and mutation-dependent manner

  • Treatment with agents like oxaliplatin can induce expression changes

  • Include appropriate treatment controls when studying regulated expression

What are the optimal protocols for C4BPA detection in Western blot applications?

For successful Western blot detection of C4BPA, researchers should follow these optimized protocols:

Sample Preparation:

• Human plasma/serum samples are excellent positive controls

• For cell/tissue lysates, ensure complete lysis to release both membrane-associated and intracellular C4BPA

• Include protease inhibitors to prevent degradation

Gel Electrophoresis Parameters:

• Use 8-10% SDS-PAGE gels for optimal resolution around 70 kDa

• Load 20 μg of human plasma lysate (as referenced in the commercial antibody protocols)

• Include molecular weight markers that span the 50-100 kDa range

Antibody Selection and Dilution:

• Primary antibody recommendations:

  • Rabbit monoclonal antibodies: 1/1000 to 1/10000 dilution

  • Rabbit polyclonal antibodies: 1/500 to 1/1000 dilution

• Secondary antibody: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Expected Results:

• Predicted band size: 67 kDa

• Observed band size: 70 kDa

• This slight discrepancy is common due to post-translational modifications

Special Considerations:

• When studying mutations that affect expression levels (e.g., 182C>T, 563G>A), include appropriate controls

• For treatment studies (e.g., oxaliplatin), compare treated vs. untreated samples

• Consider both extracellular and intracellular pools when interpreting results

How can I implement C4BPA as a biomarker in cancer research studies?

Implementing C4BPA as a cancer biomarker requires careful methodological considerations:

Selection of Detection Method:

• Tissue Analysis (IHC):

  • Follow optimized IHC protocols as described in section 2.1

  • Use standardized scoring system (e.g., comparison to internal control)

  • Consider automated image analysis for quantification

• Serum/Plasma Analysis (ELISA):

  • Use validated ELISA kits specific for C4BPA (range: 0.469 - 30 ng/ml)

  • Include appropriate controls and standards

  • Consider batch effects when analyzing multiple samples

Cancer-Specific Considerations:

Based on research findings, tailor your approach to the specific cancer type:

• Breast Cancer:

  • Focus on decreased expression compared to normal tissue

  • Correlate with prognostic indicators and survival data

  • Pay special attention to Luminal A subtype, where low C4BPA expression has stronger prognostic significance

• Pancreatic Ductal Adenocarcinoma:

  • Consider serum C4BPA as an early detection marker

  • Analyze stromal C4BPA in relation to CD8+ tumor-infiltrating lymphocytes

  • Investigate potential therapeutic applications through C4BPA-CD40 interaction

• Colorectal Cancer:

  • Screen for specific mutations (e.g., 182C>T, 563G>A)

  • Assess intracellular C4BPA levels and NF-κB pathway activation

  • Study response to therapies like oxaliplatin in relation to C4BPA status

Statistical Analysis Framework:

Reporting Guidelines:

• Follow TRIPOD reporting checklist for biomarker studies

• Clearly document antibody validation, cut-off determination, and statistical methods

• Consider independent validation cohorts for biomarker confirmation

What emerging applications of C4BPA antibodies show promise in cancer immunotherapy research?

Research suggests several promising directions for C4BPA in cancer immunotherapy:

C4BPA-CD40 Axis as Therapeutic Target:

• C4BPA binds to CD40, stimulating B cell proliferation and T cell activation

• In PDAC models, mC4BPA peptide (30 amino acids from C-terminus) increased CD8+ tumor-infiltrating lymphocytes

• Combination approaches with C4BPA peptides and conventional chemotherapy show enhanced efficacy

Potential for Combination Therapies:

• Research demonstrates that combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis

• Preclinical experiments have assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in mouse models

• C4BPA antibodies could help monitor treatment response in such combination approaches

Intracellular C4BPA as a Target:

• The role of intracellular C4BPA in regulating NF-κB-dependent apoptosis suggests potential for targeted therapies

• C4BPA mutations found in colorectal cancer alter protein levels and response to treatment

• Understanding mutation-specific effects could enable personalized therapeutic approaches

Biomarker Development:

• Low C4BPA expression correlates with poor prognosis in breast cancer

• C4BPA has potential as an early detection marker for PDAC

• Antibody-based assays for C4BPA could improve patient stratification and treatment selection

How might C4BPA research contribute to our understanding of immune regulation in disease?

C4BPA sits at a critical intersection between complement regulation, adaptive immunity, and cancer biology:

Bridge Between Innate and Adaptive Immunity:

• C4BPA regulates the classical complement pathway (innate immunity)

• It also interacts with CD40, stimulating T and B cell responses (adaptive immunity)

• This dual role provides insights into cross-talk between immune system components

Tumor Microenvironment Modulation:

• C4BPA expression correlates with immune cell infiltration patterns

• Strong positive correlations with B cells, neutrophils, pDCs, and CD8+ T cells

• Negative correlation with Th2 cells suggests complex immunoregulatory functions

Stress-Responsive Immune Regulation:

• Intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner

• This suggests a role in cellular stress responses that may impact immune recognition

• Understanding these mechanisms could reveal new approaches for immunomodulation

Beyond Cancer Applications:

• Research in bovine mammary epithelial cells shows C4BPA involvement in inflammation and milk fat synthesis

• C4BPA has binding sites for heparin, C-reactive protein, and CD40, key mediators in inflammation and blood coagulation

• These interactions suggest roles in infectious diseases, autoimmunity, and metabolic disorders