CFHR5 Antibody, Biotin conjugated

What is CFHR5 and why is it important in complement regulation?

CFHR5 (Complement Factor H-Related protein 5) is a 65 kDa plasma protein synthesized primarily by the liver. It consists of nine short consensus repeat (SCR) domains, making it the longest protein in the CFHR family. Its importance stems from its role in complement regulation, where the dimerized forms have avidity for tissue-bound complement fragments and efficiently compete with the physiological complement inhibitor CFH . CFHR5 forms homodimers through its two N-terminal domains SCR-1/2, classifying it as a factor H family I histone, along with CFHR1 and CFHR2 . Despite its functional significance, CFHR5 circulates at relatively low concentrations of approximately 2.5-3.4 μg/ml in healthy individuals, as determined by quantitative mass spectrometry and immunoassays .

What are the key structural and functional domains of CFHR5 relevant to antibody design?

CFHR5 contains 9 SCR domains (SCR1-9) with distinct functional properties. The N-terminal domains SCR1 and SCR2 are homologous to the first two SCR domains of CFHR1 and CFHR2 and are critical for dimerization . When designing antibodies, researchers should consider:

• Antibodies targeting amino acids 344-569 (C-terminal region) are effective for multiple applications including Western blotting, ELISA, and immunohistochemistry

• The protein maintains specific tertiary structures in non-reducing conditions that may be essential for certain antibody binding epitopes

• CFHR5's glycosylation pattern affects its apparent molecular weight, with de-glycosylated forms appearing at approximately 64 kDa and glycosylated forms at 70 kDa

Understanding these structural elements is crucial when selecting antibodies for specific experimental applications.

How can I verify the specificity of anti-CFHR5 antibodies?

Verifying specificity is crucial due to the high homology between CFHR family members. Recommended verification methods include:

• Immunocapture-mass spectrometry (IC-MS): This technique identified CFHR5 as the predominant protein captured by certain antibodies with z-scores >5 and multiple Peptide Spectrum Matches (≥21 PSM)

• Dot blot analysis with recombinant proteins: Test cross-reactivity against other CFHR family members (CFHR1-4) using purified recombinant fragments

• Dual binder assays: Develop assays using different combinations of capture and detection antibodies that target distinct epitopes of CFHR5

• Western blotting under different conditions: Compare results under reducing versus non-reducing conditions to identify conformation-dependent epitopes

• Deglycosylation experiments: Test antibody recognition of both glycosylated and deglycosylated forms of CFHR5

Specificity verification is particularly important since some antibodies initially developed against other targets (e.g., SULF1) have been found to predominantly bind CFHR5 in plasma .

What are the optimal applications for biotin-conjugated anti-CFHR5 antibodies?

Biotin-conjugated anti-CFHR5 antibodies are particularly valuable for specific research applications:

The goat polyclonal biotinylated anti-CFHR5 antibody (such as R&D Systems BAF3845) has been successfully used to detect recombinant human CFHR5 (Glu19-Glu569) and is particularly effective in Western blot applications at a concentration of 0.1 μg/mL .

How should I optimize protocols for detecting CFHR5 in different sample types?

Protocol optimization varies by sample type:

Plasma/Serum Samples:

• Use appropriate dilution (typically 1:100 to 1:500) to account for the relatively low physiological concentration of CFHR5 (2.5-3.4 μg/ml)

• Include specific blocking agents (5% non-fat dry milk in TBST) to minimize background

• Consider deglycosylation treatment to distinguish between glycosylated (70 kDa) and de-glycosylated (64 kDa) forms

Tissue Samples:

• For immunohistochemistry on paraffin-embedded tissues, use EDTA-based antigen retrieval (pH 8.0) for 15 minutes

• Optimize antibody dilution (1:500 for IHC-P with polyclonal antibodies)

• Validate specificity using appropriate positive control tissues (e.g., liver, as CFHR5 is liver-synthesized)

Cell Lysates:

• For HepG2 and other hepatic cell lines, use antibody dilutions of approximately 1:3000 for Western blot

• Target predicted band size of 64 kDa, but expect variations based on post-translational modifications

What are the most effective dual-binder assay configurations for quantifying CFHR5?

Dual-binder assays are crucial for specific and sensitive CFHR5 quantification:

• Validated Configuration Options:

  • Capture: HPA072446 + Detection: MAB3845 monoclonal antibody (most effective for absolute quantification)

  • Capture: HPA073894 + Detection: MAB3845 monoclonal antibody

  • Capture: HPA059937 + Detection: MAB3845 monoclonal antibody

• Performance Metrics:
  These configurations demonstrate statistically significant differentiation between case and control samples in venous thromboembolism studies (p=0.0001, 0.0021, and 0.0006 respectively) .

• Standardization:

  • Use recombinant CFHR5 protein as a standard for absolute quantification

  • Expected concentrations in control plasma: 2467-2842 ng/ml (consistent with mass spectrometry estimates of ~1900 ng/ml)

• Cross-reactivity Minimization:

  • Combining different epitope-targeting antibodies reduces potential cross-reactivity with other CFHR family members

  • Verification by IC-MS confirms specific targeting of CFHR5

How is CFHR5 implicated in venous thromboembolism (VTE) research?

Research has established significant associations between CFHR5 levels and VTE:

• Concentration Differences:
  Mean CFHR5 concentrations were significantly higher in VTE patients (3428 ± 774 ng/ml) compared to controls (2842 ± 756 ng/ml)

• Validation Across Multiple Studies:
  The association between elevated CFHR5 and VTE risk has been replicated in independent cohorts, including VEBIOS ER, VEBIOS Coagulation, DFW-VTE, and FARIVE studies

• Methodological Considerations:

  • Use absolute quantification with dual-binder assays and recombinant standards

  • Account for potential confounding factors in study design

  • Consider temporal relationship between CFHR5 elevation and thrombotic events

• Research Applications:

  • Biotin-conjugated anti-CFHR5 antibodies are valuable tools for developing high-sensitivity assays for VTE risk stratification

  • Consider using paired antibodies targeting different epitopes to enhance specificity in clinical studies

What is the relationship between CFHR5 genetic variants and age-related macular degeneration (AMD)?

Genetic studies have revealed important connections between CFHR5 variants and AMD risk:

• Protective Genetic Variants:

  • CFHR5 frameshift mutation (CFHR5fs) and CFHR5 p.Gly278Ser variants are associated with reduced AMD risk

  • The protective effect is independent of and adds to other protective variation at the CFH locus

• Protein Level Effects:

  • Carriers of protective variants show significant reduction in FHR-2, FHR-4, and FHR-5 serum levels

  • This reduction correlates with decreased AMD risk in population studies

• Haplotype Analysis:
  Five risk groups were defined based on CFH regional haplotypes, with individuals carrying only risk alleles showing the highest probability of AMD diagnosis (OR=1.67)

• Research Applications:

  • Biotin-conjugated antibodies can be used to assess CFHR5 protein levels in relation to genetic variants

  • Assays measuring CFHR5 concentration may serve as functional readouts of genetic risk status

How can antibody-based CFHR5 detection contribute to CFHR5 nephropathy research?

CFHR5 nephropathy represents an important research area where specialized antibodies are critical:

• Detection in Renal Tissues:

  • Biotin-conjugated antibodies offer enhanced sensitivity for detecting glomerular CFHR5 deposits

  • Immunohistochemical methods require optimization specific to kidney tissues

• Disease Monitoring:

  • Quantification of plasma CFHR5 using dual-binder assays may correlate with disease activity

  • Serial measurements could potentially track therapeutic responses

• Variant-Specific Detection:

  • Designing epitope-specific antibodies may distinguish between normal and mutant CFHR5 forms

  • This approach could facilitate screening and diagnosis of CFHR5 nephropathy

• Methodological Considerations:

  • Use kidney tissue lysates at 20 μg concentration for Western blot analysis

  • Consider the impact of sample preparation methods on CFHR5 detection in renal specimens

What strategies can resolve cross-reactivity issues between CFHR family members?

Cross-reactivity is a significant challenge due to high sequence homology between CFHR proteins:

• Epitope Selection:

  • Target unique regions of CFHR5 not shared with other CFHR proteins

  • Antibodies targeting AA 344-569 have demonstrated specificity

• Validation Methods:

  • Perform dot blot analysis against all recombinant CFHR family members (CFHR1-5)

  • Use immunocapture-mass spectrometry to confirm antibody targets

• Negative Controls:

  • Include human heart tissue as a negative control (confirmed by Human Integrated Protein Expression Database-GeneCards)

  • Test antibody response against recombinant CFHR1-4 proteins to verify specificity

• Optimization Example:
  An anti-CFHR5 antibody [EPR25711-28] was verified not to cross-react with human CFHR1, CFHR2, CFHR3, or CFHR4 when tested at 10 ng loading of each recombinant fragment

How do denaturation and glycosylation affect CFHR5 antibody binding?

Protein conformation and post-translational modifications significantly impact antibody recognition:

• Denaturation Effects:

  • Some antibodies only recognize CFHR5 under non-reducing conditions, suggesting epitope dependence on tertiary structure

  • Western blot analysis revealed that certain antibodies detect monomeric CFHR5 under non-reducing but not reducing conditions

• Glycosylation Impact:

  • CFHR5 exists in both glycosylated (~70 kDa) and de-glycosylated (~64 kDa) forms

  • Deglycosylation treatment of human plasma samples (using Protein Deglycosylation Mix II) alters the apparent molecular weight in Western blots

  • Some epitopes may be masked or exposed depending on glycosylation status

• Methodological Recommendations:

  • Always include both reducing and non-reducing conditions when characterizing new antibodies

  • Consider parallel analysis of deglycosylated samples for comprehensive epitope mapping

  • Use recombinant standards with defined glycosylation status for quantification assays

What are the optimal storage and reconstitution conditions for maintaining biotin-conjugated CFHR5 antibody activity?

Proper handling is essential for maintaining antibody performance:

• Storage Guidelines:

  • Store lyophilized antibody at -20°C to -70°C

  • After reconstitution, store at 2-8°C for short-term (1 month) under sterile conditions

  • For long-term storage (6 months), aliquot and store at -20°C to -70°C

• Reconstitution Protocol:

  • Reconstitute at 0.2 mg/mL in sterile PBS

  • Allow complete dissolution before use

  • Avoid repeated freeze-thaw cycles by storing in single-use aliquots

• Stability Indicators:

  • Monitor for aggregation or precipitation

  • Validate activity periodically against positive controls

  • Consider including protein stabilizers (e.g., BSA) for diluted working solutions

• Performance Optimization:

  • Pre-titers for specific applications: Western Blot (0.1 μg/mL)

  • Perform antibody titration experiments to determine optimal concentration for each application

  • Validate each new lot against previous standards