CFHR5 Antibody

What is CFHR5 and what role does it play in the complement system?

CFHR5 (Complement Factor H-Related protein 5) is a member of the complement Factor H protein family that plays a role in complement regulation. The dimerized forms of CFHR5 have avidity for tissue-bound complement fragments and efficiently compete with the physiological complement inhibitor CFH . While initially identified as a universal component of complement deposits, CFHR5 is synthesized primarily in the liver and has been detected in glomerular immune deposits . Its pattern of deposits resembles other complement components, suggesting its involvement in complement activation and regulation pathways .

CFHR5 exhibits similar characteristics to Factor H, including heparin binding, CRP binding, and lipoprotein association capabilities . Additionally, weak Factor I-dependent cofactor activity for C3b cleavage has been observed with CFHR5 . These properties collectively suggest that CFHR5 participates in fine-tuning complement activation, particularly at the tissue level.

How is CFHR5 structurally organized and how does this impact its function?

CFHR5 is the longest protein in the CFHR family, consisting of 9 short consensus repeat (SCR) domains with a complete protein length of 551 amino acids . Its modular organization is functionally significant:

• SCR1 and SCR2 domains at the N-terminus form the dimerization interface

• SCR1 and SCR2 are homologous to the first two SCR domains of CFHR1 and CFHR2

• The full protein forms homodimers through its two N-terminal domains, classifying it as a Factor H family I protein along with CFHR1 and CFHR2

Despite its impressive size, CFHR5 circulates at relatively low concentrations in blood (approximately 3-6 μg/ml), making it the least abundant of the CFHR proteins .

What types of CFHR5 antibodies are available and how should they be selected for specific applications?

Several types of CFHR5 antibodies are commercially available with varying specifications:

When selecting a CFHR5 antibody, researchers should consider:

• The specific application (IHC, WB, ELISA, etc.)

• Required specificity (potential cross-reactivity with other CFHR family members)

• Format needs (capture vs. detection antibody)

• Validation data available for the specific application

How can researchers validate CFHR5 antibody specificity to avoid cross-reactivity with other Factor H family proteins?

Cross-reactivity is a significant concern when working with CFHR family proteins due to high sequence homology. To validate CFHR5 antibody specificity:

• Dot Blot Analysis: Use recombinant proteins from the entire CFHR family. For example, ab305247 has been validated using dot blot analysis against His-tagged human CFHR1-5 recombinant fragments, demonstrating no cross-reactivity with CFHR1-4 .

• Western Blot Confirmation: Validate using both recombinant proteins and natural sources such as HepG2 cell lysate, which expresses CFHR5 .

• Immunoprecipitation-Mass Spectrometry (IP-MS): This approach can definitively identify captured proteins. When the antibody HPA059937 was validated using IP-MS, CFHR5 was confirmed as the predominant captured protein in plasma .

• Dual Binder Assays: Develop assays using multiple antibodies against different epitopes of CFHR5, such as the system using MAB3845 as detection antibody combined with different capture antibodies (HPA059937, HPA073894, HPA072446) .

• Specificity Control: When developing an ELISA, confirm that the monoclonal anti-FHR-5 used as capture antibody does not detect FH or any FHR other than FHR-5 through Western blot .

What is the recommended protocol for developing a robust ELISA to measure FHR-5 levels in serum samples?

Based on validated methods from recent research, the following protocol has proven effective for FHR-5 quantification in serum:

Sandwich ELISA Protocol for FHR-5 Measurement:

• Coating: Coat microtiter ELISA plates with 1 μg/ml monoclonal mouse anti-human FHR-5 (IgG1, clone #390513, R&D Systems) in phosphate-buffered saline (PBS) overnight at 4°C .

• Blocking: Block plates with PBS containing 2% bovine serum albumin (BSA) for 1 hour at room temperature .

• Sample Preparation: Dilute serum 1:100 in PBS containing 1% BSA and 0.05% Tween-20 .

• Incubation: Add diluted samples to plates and incubate for 1 hour at room temperature .

• Detection: Use polyclonal goat anti-human FHR-5 IgG (Cat. number: AF3845, R&D Systems) for detection .

• Standard Curve: Prepare standard curve using 2-fold dilution series of recombinant human FHR-5 protein (R&D Systems) .

• Quality Control: Determine inter-assay and intra-assay variations (reported as 11.8% and 7.8%, respectively in published methods) .

• Specificity Validation: Confirm specificity using Western blot to ensure the monoclonal anti-FHR-5 capture antibody does not detect FH or any FHR other than FHR-5 .

How can researchers accurately measure C3b-binding ability of CFHR5 variants?

To assess functional differences in C3b-binding capacity of CFHR5 variants, two complementary approaches have been validated:

ELISA-Based Method:

• Coat microtiter plates with 5 μg/ml C3b fragment (Merck) overnight at 4°C .

• Block with DPBS containing 2% BSA for 1 hour at room temperature .

• Dilute serum samples 1:4 in DPBS containing 1% BSA and 0.05% Tween-20, along with a dilution series (3.9–250 ng/ml) of recombinant human FHR-5 as standard .

• Apply samples to the plate and incubate for 1 hour at 37°C .

• Detect binding with monoclonal mouse anti-human FHR-5 followed by HRP-labeled secondary antibody .

• Develop with TMB substrate and measure absorbance at 450/620 nm .

Surface Plasmon Resonance (SPR) Method:
This approach provides more detailed binding kinetics information, including:

For example, when analyzing the C3b binding of FHR-5 G278S variant compared to wild-type, SPR analysis revealed a KD value of 2.05 × 10−5 M for C3b–FHR-5 G278S versus 5.26 × 10-6 M for wild-type, demonstrating quantitatively weaker binding .

How are CFHR5 genetic variations linked to glomerular diseases?

CFHR5 genetic variations have been extensively studied in relation to kidney diseases, particularly C3 glomerulopathies. Key findings include:

What experimental approaches are most effective for analyzing CFHR5 function in disease models?

Multiple complementary approaches have proven valuable for studying CFHR5 in disease contexts:

• Recombinant Protein Expression and Purification:

  • Express wild-type and variant CFHR5 proteins to compare functional differences

  • Study specific mutations of interest using site-directed mutagenesis

• Binding Assays:

  • ELISA-based quantification of C3b binding capacity

  • Surface plasmon resonance (SPR) for detailed kinetic binding parameters

  • Competitive binding assays with Factor H

• Serum Proteomics:

  • Measure serum FHR-5 levels in patient cohorts using validated ELISA methods

  • Analyze correlation between genetic variants and circulating protein levels

  • Use high-throughput proteomic platforms like SomaScan for measuring multiple complement proteins simultaneously

• Genetic Association Studies:

  • Sequence CFHR5 gene in patient cohorts with relevant kidney diseases

  • Analyze variant frequency and association with disease phenotypes

  • Study haplotypes across the CFH/CFHR gene cluster

• Functional Complement Assays:

  • Measure total complement pathway activity using hemolytic titration tests

  • Quantify specific complement components (C3, C4, Factor B, Factor D)

  • Assess complement activation products (C3a, Bb, C4d, sC5b-9)

How can researchers distinguish between different CFHR5 variants in experimental settings?

Distinguishing between CFHR5 variants requires a combination of molecular and biochemical techniques:

• Genetic Analysis:

  • Direct bidirectional DNA sequencing of the entire CFHR5 coding region

  • Use of carefully designed primers to detect specific variants or duplications

  • Next-generation sequencing for comprehensive variant detection

• Protein Characterization:

  • Western blot analysis may detect size differences for some variants (particularly duplications or truncations)

  • Mass spectrometry can identify specific amino acid changes in purified protein

  • Isoelectric focusing can distinguish variants with changed charge properties

• Functional Differentiation:

  • C3b binding assays reveal functional differences between variants

  • Competitive binding assays with Factor H

  • Cell-based complement activation assays

• Epitope-Specific Antibodies:

  • Development of antibodies recognizing specific variant epitopes

  • Use of multiple antibodies targeting different regions of the protein

What considerations are important when studying interactions between CFHR5 and other complement proteins?

The study of CFHR5's interactions with other complement components requires careful experimental design:

• Purity and Specificity:

  • Ensure recombinant proteins are highly pure to avoid contamination with other complement factors

  • Validate antibody specificity using dot blot or Western blot against all Factor H family proteins

• Physiological Relevance:

  • Consider the natural concentration of CFHR5 in circulation (3-6 μg/ml)

  • Account for the presence of other FHR proteins that may compete for binding sites

  • Study interactions in physiologically relevant buffers and conditions

• Multimeric State:

  • Consider the dimeric state of CFHR5 in solution

  • Study both monomeric and dimeric forms where possible

  • Assess how dimerization affects binding properties

• Interaction Dynamics:

  • Use SPR for detailed kinetic analysis of binding parameters

  • Consider the impact of local concentrations in tissues versus circulation

  • Assess pH and ionic strength effects on interactions

• Competitive Interactions:

  • Design experiments to assess competition between CFHR5 and Factor H

  • Investigate how CFHR5 variants affect competition with Factor H

  • Consider the role of other FHR proteins in competition for binding sites

What are common challenges when working with CFHR5 antibodies and how can they be addressed?

Researchers working with CFHR5 antibodies commonly encounter several technical challenges:

• Cross-reactivity with other FHR proteins:

  • Solution: Use validated antibodies with demonstrated specificity, such as those tested by dot blot against all FHR proteins

  • Perform pre-absorption steps with recombinant FHR1-4 proteins to remove cross-reactive antibodies

  • Validate specificity by Western blot or immunoprecipitation followed by mass spectrometry

• Low circulating levels:

  • Solution: Optimize sample preparation with appropriate concentration steps

  • Use high-sensitivity detection methods

  • Consider enrichment techniques prior to analysis

• Variable glycosylation:

  • Solution: Account for potential differences in mobility on SDS-PAGE

  • Include deglycosylation controls when necessary

  • Consider using recombinant proteins expressed in different systems

• Interference from other serum components:

  • Solution: Optimize blocking conditions (2% BSA has been validated)

  • Include detergents (0.05% Tween-20) in sample diluents

  • Consider sample purification steps before analysis

How can researchers validate novel findings related to CFHR5 function or disease associations?

To ensure robustness of novel CFHR5 findings, researchers should implement multi-level validation:

• Genetic Validation:

  • Confirm variants in independent cohorts

  • Establish statistical significance with appropriate power

  • Consider population stratification and ancestry

• Protein Expression Validation:

  • Correlate genetic findings with protein levels in serum

  • Use multiple antibodies targeting different epitopes

  • Include appropriate controls (e.g., samples from individuals with known CFHR5 deficiency)

• Functional Validation:

  • Test multiple functional aspects (C3b binding, complement regulation)

  • Compare recombinant proteins with native proteins from patient samples

  • Use both cell-free and cell-based systems

• Clinical Correlation:

  • Associate findings with disease parameters and outcomes

  • Perform longitudinal studies where possible

  • Consider confounding factors (e.g., other genetic variants, environmental factors)

• Methodological Controls:

  • Include positive and negative controls in all assays

  • Ensure appropriate statistical analysis

  • Validate findings using different technological approaches (e.g., ELISA, SPR, functional assays)