C4BPB Antibody

What is C4BPB and what is its significance in the complement system?

C4BPB (Complement Component 4 Binding Protein Beta) is a plasma glycoprotein that functions as a critical regulator of the classical and lectin pathways of complement activation. The beta chain of C4BP is encoded by the C4BPB gene located in chromosome 1q32 within the regulator of complement activation gene cluster .

C4BPB forms part of a multimeric protein complex that inhibits uncontrolled complement activation by:

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

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

• Specifically binding to protein S, which has implications in coagulation regulation

These mechanisms collectively reduce bacterial opsonization and phagocytosis, playing a role in immune homeostasis .

What are the structural properties of C4BPB antibodies available for research?

Most commercial C4BPB antibodies are available in several formats:

• Host species and types: Predominantly rabbit polyclonal (like PA5-76914) and rabbit monoclonal (like EPR17101) antibodies

• Purification methods: Typically affinity-purified using epitope-specific immunogens with >95% purity (by SDS-PAGE)

• Storage conditions: Most stable at -20°C for up to one year in PBS buffer with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Formats: Available as unconjugated antibodies or conjugation-ready formats for fluorochromes, metal isotopes, oligonucleotides, and enzymes

Research-grade antibodies typically recognize human C4BPB with a molecular weight of approximately 45 kDa, though the calculated molecular weight is around 28 kDa (251 amino acids) .

What are the validated applications for C4BPB antibodies in experimental protocols?

C4BPB antibodies have been validated for multiple research applications with specific dilution recommendations:

For optimal results in IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative .

How should researchers optimize Western blot protocols specifically for C4BPB detection?

For optimal Western blot detection of C4BPB:

• Sample preparation: Human plasma, hepatocyte cell lines, or liver tissue lysates provide reliable detection

• Loading controls: Use 5% non-fat dry milk in TBST as blocking buffer

• Antibody concentration: Start with 1:1000 dilution for primary antibody incubation

• Molecular weight identification: Look for bands at approximately 45 kDa, which is the observed molecular weight for C4BPB, rather than the calculated 28 kDa

• Validation: The observed molecular weight is consistent with literature reports (PMID: 2300577)

Note that non-specific bands may appear, and optimization of blocking conditions and antibody concentration may be required for different tissue types .

How do C4BPB antibodies help distinguish between different C4BP isoforms?

C4BP exists in three main isoforms: α7β1, α7β0, and α6β1, based on the number of identical α-chains (6 or 7) and the presence/absence of a single β-chain . Researchers studying these isoforms should consider:

• Antibodies targeting the β-chain (C4BPB) are crucial for distinguishing β-chain containing isoforms (α7β1, α6β1) from those lacking it (α7β0)

• The α7β0 isoform represents approximately 17% of C4BP molecules in normal plasma

• Specific monoclonal antibodies against different epitopes (e.g., those recognizing the 48K or 160K fragments) can be used to study functional domains

• MRM-MS (Multiple Reaction Monitoring-Mass Spectrometry) techniques can provide more precise quantification of specific isoforms

Understanding these isoforms is critical as the amount of β-chain-containing isoforms serves as a surrogate marker for free protein S (fPS) levels, relevant in coagulation studies .

What functional domains of C4BPB can be targeted by different antibodies, and how does this affect experimental outcomes?

Research with monoclonal antibodies has identified specific functional domains in C4BPB:

• 48K fragment domain: Some monoclonal antibodies (e.g., TK3) specifically target this domain and can:

  • Block C4BP binding to cell-bound C4b

  • Inhibit C4BP's cofactor activity for C3b/C4b inactivator

  • Prevent acceleration of C2a decay from the C4b,2a complex

• 160K fragment domain: Antibodies targeting this region typically don't affect the functional activities listed above

• Other epitopes: Some antibodies recognize intact C4BP but not specific fragments, suggesting they target conformational epitopes or junction regions

For functional studies, researchers should select antibodies targeting specific domains based on their experimental goals. The 48K fragment appears to be the functionally active site of C4BP, though its activity is approximately 200 times weaker than intact C4BP on a molar basis .

How can C4BPB antibodies be used to investigate microbial evasion of complement?

Certain bacterial pathogens, particularly Streptococcal strains, exploit C4BP to evade complement activation. When researching this mechanism:

• Experimental approach: Use C4BPB antibodies to:

  • Block C4BP binding to bacterial surface proteins

  • Visualize C4BP recruitment to bacterial surfaces

  • Study competition between bacterial ligands and natural C4b for C4BP binding sites

• Research innovations: Novel C4BP-IgM fusion proteins have been developed that can outcompete native C4BP for binding to gonococci, increasing bacterial susceptibility to complement-mediated killing

• Antibiotic synergy: C4BP-IgM in conjunction with normal human serum increased the sensitivity of gonococci to antibiotics and restored sensitivity in azithromycin-resistant strains by promoting complement activation, pore formation, and antibiotic entry

This area represents a promising avenue for investigating new approaches to combat antimicrobial resistance.

What is the significance of C4BPB genetic variants in thrombotic disorders, and how can researchers study these associations?

Genome-wide association studies have identified SNPs in the C4BPB/C4BPA gene cluster associated with venous thrombosis risk. Key considerations for researchers investigating these associations include:

Interestingly, these genetic variants affect C4BP isoform distribution but do not significantly impact free Protein S (fPS) or total PS levels, suggesting a protein S-independent mechanism for thrombosis risk .

Researchers investigating these associations should:

• Consider genotyping these SNPs in thrombosis case-control studies

• Measure plasma levels of different C4BP isoforms using specific antibodies

• Conduct functional studies to understand how altered isoform distribution affects complement regulation and coagulation

How is C4BPB being investigated in neuropsychiatric and neuroinflammatory conditions?

Recent research has begun exploring the relationship between C4BP levels and neurological conditions:

• White matter tract integrity: Studies have examined correlations between serum C4BP levels and white matter tract integrity in major depressive disorder (MDD), representing the first investigations in this area

• Methodology for CNS studies:

  • Plasma sample preparation for C4BP detection

  • Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS) for confirmational studies

  • Quantification of serum protein levels

  • Correlation with MRI data acquisition and analysis

• Technical challenges: When studying C4BPB in neurological contexts, researchers must consider blood-brain barrier permeability, local CNS production versus systemic levels, and potential confounding by inflammatory conditions

This represents an emerging field connecting complement regulation to neurological health and disease.

What are the complement-independent functions of C4BPB that researchers are currently investigating?

Beyond its canonical role in complement inhibition, C4BPB/C4BP has multiple complement-independent functions that represent active areas of research:

• Viral pathogenesis modulation: C4BP has been found to bind H1N1 influenza A virus, downregulating IL-12, TNF-α, and NFκB levels in challenged lung epithelial cells. Interestingly, C4BP produces opposing effects when binding H3N2 IAV subtype, promoting viral endocytosis and upregulating proinflammatory cytokine production

• Cell survival regulation: C4BP plays roles in promoting apoptotic cell death while also having complement-independent functions in promoting cell survival in other contexts

• Autoimmunity protection: Evidence suggests C4BP helps protect against autoimmune damage through mechanisms beyond complement inhibition

• Cancer research applications: C4BP is being studied for its role in controlling excessive inflammation in cancer and chronic diseases

These diverse functions highlight the importance of using specific antibodies to study the multifaceted roles of C4BPB in different biological contexts.

What are common pitfalls in C4BPB antibody experiments and how can researchers overcome them?

When working with C4BPB antibodies, researchers may encounter several challenges:

• Molecular weight discrepancies: The observed molecular weight (45 kDa) differs from the calculated weight (28 kDa). Researchers should verify antibody specificity through positive controls like human plasma or HeLa cells

• Sample preparation considerations:

  • Human plasma provides the most reliable detection

  • For cell lines, hepatocyte-derived lines (HepG2, HeLa) show better expression

  • When using tissue samples, liver tissue provides the strongest signal

• Species cross-reactivity limitations:

  • Most antibodies are validated for human samples

  • Mouse reactivity is confirmed for some antibodies

  • Rat reactivity may be present but requires validation

  • Other species should be tested empirically

• Storage and stability issues: Store antibodies at -20°C; they remain stable for one year after shipment. For 20μl sizes containing 0.1% BSA, aliquoting is unnecessary for -20°C storage

How can researchers effectively validate the specificity of C4BPB antibodies?

Thorough validation is essential for reliable C4BPB antibody experiments:

• Positive controls: Use human plasma tissue, HeLa cells, or mouse ovary tissue for Western blot validation

• Multiple application validation: Confirm antibody performance across different applications:

  • For Western blot: Human plasma provides reliable detection

  • For IHC: Human liver tissue with TE buffer pH 9.0 for antigen retrieval

  • For IF/ICC: HepG2 cells show consistent staining

• Functional blocking experiments: Validate antibody specificity by testing its ability to block C4BP binding to C4b or other known ligands

• Knockdown/knockout verification: Where possible, use C4BPB knockdown or knockout samples to confirm antibody specificity

• Epitope mapping: Consider using antibodies that target specific functional domains (like the 48K fragment) to correlate with biological activity