CFHR5 Antibody, HRP conjugated

What is CFHR5 and why is it significant for immunological research?

CFHR5 is a 65-kDa plasma glycoprotein produced in the liver, with a reported serum concentration of approximately 3-6 μg/ml. It consists of nine CCP domains that are related to CCPs 6-7, CCPs 10-14, and CCPs 19-20 of Factor H (FH) . CFHR5 has significant research importance because it was originally isolated from human glomerular complement deposits and has been detected in glomerular immune deposits in several kidney diseases, including membranous nephropathy, IgA nephropathy, lupus nephritis, focal glomerular sclerosis, and postinfectious glomerulonephritis . The protein can bind to multiple targets including heparin, C-reactive protein, pentraxin-3, and extracellular matrix . CFHR5 may enhance complement activation by interfering with the complement-inhibiting function of FH, enhancing C1q binding, and directly activating complement, thereby potentially contributing to glomerular disease .

What detection methods work best for CFHR5 proteins in research samples?

Multiple detection methods have demonstrated efficacy for CFHR5 research:

• ELISA: Custom ELISA systems using monoclonal or polyclonal antibodies against CFHR5 have been developed for quantitative detection. A validated approach uses monoclonal mouse anti-human FHR-5 as capture antibody and polyclonal goat anti-human FHR-5 IgG for detection, followed by HRP-labeled secondary antibodies . The specificity of such ELISAs should be confirmed by Western blot to ensure the capture antibody doesn't detect FH or other FHR proteins .

• Western Blotting: Particularly effective when using polyclonal antibodies targeting specific amino acid regions of CFHR5. This technique can distinguish between native and recombinant forms, as well as detect structural changes under reducing versus non-reducing conditions .

• Surface Plasmon Resonance (SPR): This label-free technique enables analysis of CFHR5 binding kinetics to its targets such as C3b. Typically, recombinant CFHR5 variants are immobilized on a biosensor chip using amine coupling methods, and analytes are flowed across at varying concentrations .

• Immunoprecipitation followed by Mass Spectrometry (IP-MS): This approach can definitively identify CFHR5 in complex samples and verify antibody specificity .

How should CFHR5 antibody specificity be validated?

Validating CFHR5 antibody specificity requires a multi-tiered approach:

• Cross-reactivity testing: CFHR5 antibodies should be tested against other FH family proteins due to high sequence homology. For example, the monoclonal anti-FHR-5 used as capture antibody in ELISA systems should be confirmed not to detect FH or any FHR other than FHR-5 .

• Western blot analysis: Test antibody recognition under both reducing and non-reducing conditions. Some antibodies may recognize only specific conformational epitopes. For instance, the anti-SULF1 HPA059937 antibody was found to detect monomeric recombinant CFHR5 under non-reducing conditions but not reducing conditions, suggesting binding to an epitope created by the tertiary folded structure of CFHR5 .

• Dual binder assays: Using multiple antibodies targeting different epitopes of CFHR5 can confirm specificity. For example, researchers have developed dual binder assays using a commercial monoclonal antibody against CFHR5 (MAB3845) as a detection antibody, combined with different capture antibodies (HPA059937, HPA073894, or HPA072446) .

• Immunocapture mass spectrometry (IC-MS): This technique definitively identifies the protein(s) bound by an antibody in plasma or other complex samples .

What are the optimal conditions for using HRP-conjugated CFHR5 antibodies in Western blotting?

For Western blotting applications using HRP-conjugated CFHR5 antibodies, researchers should consider the following protocol optimizations:

• Sample preparation: CFHR5 exhibits different epitope accessibility under reducing versus non-reducing conditions. Some antibodies recognize CFHR5 only under non-reducing conditions due to conformational epitopes . Prepare parallel samples under both conditions when first characterizing a new antibody.

• Blocking conditions: Use 4-5% BSA in DPBS containing 0.05% Tween-20 for optimal blocking with minimal background .

• Antibody dilutions: For HRP-conjugated primary antibodies against CFHR5, typical working dilutions range from 1:1000 to 1:5000, though this must be optimized for each specific antibody.

• Detection system: TMB (3,3′,5,5′-tetramethylbenzidine) has been successfully used as a substrate for HRP-conjugated antibodies in CFHR5 detection systems .

• Washing steps: Thorough washing with DPBS containing 0.05% Tween-20 is critical between steps to reduce background signal.

• Controls: Include recombinant CFHR5 protein (such as from R&D Systems) as a positive control at known concentrations to establish detection limits and specificity .

How can CFHR5 antibodies be used to analyze CFHR5-pentraxin interactions?

CFHR5 has been shown to interact with pentraxins, particularly PTX3 and C-reactive protein. The following methodologies have been validated for studying these interactions:

• Direct binding assays: Coat microtiter plate wells with PTX3 or CRP, incubate with CFHR5 (either recombinant or from plasma), and detect bound CFHR5 using specific antibodies . For quantitative analysis, a dilution series of recombinant CFHR5 can be used to generate a standard curve.

• Competition assays: To assess competition between CFHR5 and Factor H for pentraxin binding, wells coated with PTX3 (30 nM) or CRP (87 nM) can be incubated with 50 μg/ml FH in the absence or presence of increasing concentrations of CFHR5. FH binding is then detected with an antibody that does not recognize CFHR5, such as mAb A254 .

• Serum competition studies: For more physiologically relevant conditions, heat-inactivated human serum (25%) can be used with or without added CFHR5 (0.5 μM) on PTX3 or CRP-coated surfaces .

• Functional consequence analysis: Following binding of PTX3 to CFHR5, researchers can assess downstream effects such as increased C1q binding, which suggests a role for CFHR5 in modulating complement activation on pentraxin-decorated surfaces .

What experimental approaches are effective for studying CFHR5 genetic variants?

Research into CFHR5 genetic variants, particularly those associated with CFHR5 nephropathy, can employ these validated methodologies:

• Recombinant protein production: Generate wild-type and variant CFHR5 proteins (e.g., FHR-5 G278S, FHR-5 R356H) to study functional differences .

• Comparative binding studies: Assess binding of variant CFHR5 proteins to C3b using ELISA-based methods. For example, coat microtiter plates with 5 μg/ml C3b, block with BSA, and add serial dilutions of recombinant CFHR5 variants. Detect binding with polyclonal anti-FHR-5 antibodies followed by HRP-conjugated secondary antibodies .

• Surface plasmon resonance analysis: For detailed kinetic analysis, immobilize recombinant CFHR5 variants on biosensor chips and flow C3b at different concentrations as analyte. This approach enables measurement of association and dissociation rates for different variants .

• Functional competition assays: Assess how CFHR5 variants compete with FH for binding to C3b or other ligands, providing insights into potential pathogenic mechanisms .

• Serum FHR-5 quantification: Measure FHR-5 levels in patient sera using ELISA to correlate genetic variations with protein levels. A standardized approach uses recombinant human FHR-5 protein for calibration curves .

How can cross-reactivity with other complement factors be minimized?

Cross-reactivity is a significant concern when working with CFHR5 antibodies due to the high sequence homology within the FH protein family. Several approaches can minimize this issue:

• Epitope selection: Choose antibodies targeting unique regions of CFHR5. The amino acid regions 450-569 and 374-569 have been used successfully for generating specific antibodies .

• Validation using multiple methods: Confirm antibody specificity using Western blot, ELISA, and mass spectrometry. For example, researchers confirmed that a monoclonal CFHR5 antibody (MAB3845) specifically bound CFHR5 in plasma using immunocapture-mass spectrometry analysis .

• Dual antibody approaches: Using two different antibodies targeting distinct epitopes in sandwich assays significantly increases specificity. For example, combining HPA059937, HPA073894, or HPA072446 as capture antibodies with MAB3845 as detection antibody has demonstrated high specificity for CFHR5 .

• Negative controls: Include negative controls using antibodies against other FH family proteins to confirm the absence of cross-reactivity.

• Pre-absorption: When necessary, pre-absorb antibodies with recombinant forms of potentially cross-reactive proteins.

What controls should be included in CFHR5 antibody experiments?

For rigorous CFHR5 antibody experiments, the following controls are recommended:

• Positive controls:

  • Recombinant human FHR-5 protein (such as from R&D Systems) at known concentrations

  • Plasma samples with confirmed CFHR5 expression

• Negative controls:

  • Ovalbumin or other irrelevant proteins (e.g., as used in SPR experiments)

  • Samples depleted of CFHR5

  • Isotype-matched control antibodies

• Specificity controls:

  • Other FH family proteins to confirm absence of cross-reactivity

  • CFHR5 detection under both reducing and non-reducing conditions

  • Competitive blocking with unlabeled antibodies

• Technical controls:

  • Inter-assay and intra-assay variation measurements (reported as 11.8% and 7.8%, respectively, for a validated CFHR5 ELISA)

  • Dilution series to confirm linearity and dynamic range

  • Secondary antibody-only controls to assess non-specific binding

How can researchers quantify CFHR5 levels in clinical samples?

Accurate quantification of CFHR5 in clinical samples requires validated methodologies:

• Sandwich ELISA: A validated approach uses monoclonal anti-CFHR5 antibody as capture antibody and polyclonal anti-CFHR5 for detection. The standard curve should be generated using a 2-fold dilution series of recombinant human FHR-5 protein. This method has demonstrated inter-assay and intra-assay variations of 11.8% and 7.8%, respectively .

• Sample preparation: Serum samples should be diluted (typically 1:4) in buffer containing BSA and Tween-20 to minimize matrix effects . Heat inactivation (56°C, 30 min) may be necessary for certain applications .

• Calibration: Use recombinant FHR-5 protein at concentrations ranging from 3.9-250 ng/ml to establish a reliable standard curve .

• Detection systems: For HRP-conjugated antibodies, TMB substrate provides sensitive detection with absorbance measurement at 450/620 nm .

• Verification: For clinical studies, verification of CFHR5 levels using orthogonal methods such as Western blot or mass spectrometry is recommended, especially when investigating novel associations such as venous thromboembolism .

How should researchers interpret differences in CFHR5 binding patterns?

When analyzing CFHR5 binding patterns, researchers should consider several factors that influence interpretation:

• Binding kinetics: SPR studies have demonstrated that CFHR5 binding to targets like C3b can be characterized by association and dissociation rates. These should be measured at multiple analyte concentrations (typically following a 2-fold dilution series) and analyzed using appropriate software (e.g., ProteOnManager) .

• Competitive binding: CFHR5 competes with FH for binding to targets like PTX3, CRP, and extracellular matrix. The degree of competition provides insights into the potential pathophysiological role of CFHR5. Such competition should be assessed using fixed concentrations of one protein with increasing concentrations of the competitor .

• Structural considerations: Some CFHR5 antibodies recognize epitopes that are only accessible under specific conditions (e.g., non-reducing versus reducing) . Differences in binding patterns under various conditions can provide insights into protein conformation and epitope accessibility.

• Genetic variants: Mutations in CFHR5, such as those found in CFHR5 nephropathy, can alter binding properties. Comparative analysis of wild-type and variant CFHR5 binding can reveal functional consequences of these mutations .

• Functional outcomes: Beyond mere binding, researchers should interpret how CFHR5 binding affects downstream complement activation. For example, CFHR5 binding to PTX3 increases C1q binding, suggesting enhancement of the classical pathway .

What are the established reference ranges for CFHR5 in research contexts?

Current research has established several reference parameters for CFHR5:

• Serum concentration: Normal human serum contains approximately 3-6 μg/ml of CFHR5 .

• ELISA detection range: Validated ELISA systems typically use standard curves with recombinant CFHR5 ranging from 3.9-250 ng/ml .

• Inter-assay variation: 11.8% for validated ELISA systems .

• Intra-assay variation: 7.8% for validated ELISA systems .

• Binding affinity: While specific KD values vary based on the interaction partner and experimental method, SPR studies provide quantitative measures of CFHR5 binding affinity. Both association (120 seconds) and dissociation (600 seconds) phases should be monitored for complete binding kinetics analysis .

These parameters provide important benchmarks for researchers studying CFHR5 in various disease contexts, including complement-mediated kidney diseases and potentially venous thromboembolism .

How can researchers integrate CFHR5 antibody data with genetic analysis?

Integrating CFHR5 antibody-based protein data with genetic analysis provides comprehensive insights into disease mechanisms:

• Genotype-phenotype correlation: Measure CFHR5 protein levels using specific antibodies in samples from individuals with known CFHR5 genetic variants. This approach has been applied in studying immune complex-mediated membranoproliferative glomerulonephritis and CFHR5 nephropathy .

• Functional characterization: Express recombinant CFHR5 proteins containing specific mutations (e.g., G278S, R356H) identified in patients and analyze their binding properties compared to wild-type protein using antibody-based detection systems .

• Protein expression analysis: Examine how genetic variations affect CFHR5 protein expression levels in patient samples. Antibody-based quantification can determine if variants result in increased, decreased, or functionally altered protein .

• Complement activation assessment: Use antibodies against CFHR5 and other complement components to evaluate how genetic variants affect complement regulation. This can include measurement of C3b binding, FH competition, and C1q recruitment .

• Biomarker development: Combine genetic risk profiles with antibody-based CFHR5 quantification to develop predictive biomarkers for conditions such as venous thromboembolism, where elevated plasma CFHR5 has been associated with disease risk .

How are CFHR5 antibodies being used to study novel disease associations?

Recent research has expanded the role of CFHR5 beyond kidney diseases to other conditions:

• Venous thromboembolism (VTE): CFHR5 has been identified as a VTE-associated plasma protein. Researchers used multiple antibody-based approaches to confirm CFHR5 as the predominant protein captured by antibody HPA059937 in plasma. This included developing dual binder assays with various capture antibodies and a common detection antibody (MAB3845), verifying binding specificity through immunocapture-mass spectrometry .

• Complementopathies: CFHR5 antibodies are being used to investigate complement dysregulation in various diseases. The protein's ability to enhance complement activation by competing with FH and supporting C3 convertase formation suggests its potential involvement in numerous complement-mediated conditions .

• Extracellular matrix interactions: CFHR5 binds to extracellular matrix in a dose-dependent manner and competes with FH. Antibody-based detection methods are helping researchers understand how this interaction may contribute to tissue damage in inflammatory conditions .

• Pentraxin-mediated inflammation: CFHR5's interaction with pentraxins (PTX3 and CRP) and subsequent enhancement of C1q binding suggest roles in acute phase responses. Specific antibodies enable detailed characterization of these complex interactions .

What are the latest technical advances in CFHR5 antibody development?

Recent technical advances in CFHR5 antibody development include:

• Epitope mapping: Development of antibodies targeting specific domains of CFHR5, including those recognizing amino acids 450-569, 344-569, 374-569, and 203-231 . This enables more precise analysis of CFHR5 domain functions.

• Monoclonal antibodies: Several monoclonal antibodies are now available (e.g., 3E1E10, 3A10A5) that offer high specificity and reduced batch-to-batch variation .

• Conjugated antibodies: HRP and biotin-conjugated CFHR5 antibodies eliminate the need for secondary antibodies, simplifying protocols and potentially reducing cross-reactivity .

• Dual binder assays: Development of assays using combinations of antibodies targeting different CFHR5 epitopes has significantly improved specificity and sensitivity .

• Validation methods: Advanced approaches including immunocapture-mass spectrometry are now used to definitively confirm antibody specificity in complex biological samples .

These advances continue to expand the research toolbox available for studying CFHR5 in various biological and pathological contexts.