MFAP4 Antibody

What is MFAP4 and what is its physiological significance?

MFAP4 (microfibril associated protein 4) is an extracellular matrix glycoprotein containing a fibrinogen C-terminal domain and an N-terminal integrin-binding motif. It is primarily localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), suggesting a role in tissue architecture maintenance. MFAP4 is believed to be involved in calcium-dependent cell adhesion or intercellular interactions . The protein has a calculated molecular weight of approximately 29 kDa, although it typically appears around 36 kDa in experimental conditions, likely due to post-translational modifications . MFAP4 is particularly interesting to researchers as its systemic levels are significantly elevated in patients with hepatic cirrhosis, potentially reflecting diseases with increased ECM turnover .

What tissue distribution patterns does MFAP4 exhibit?

Immunohistochemical studies have demonstrated that MFAP4 protein is predominantly localized in tissues rich in elastic fibers and within blood vessels. In human tissues, MFAP4 expression is particularly high in the elastic fibers located within blood vessels and the surrounding connective tissue . In the myocardium, MFAP4 is intensely detected in elastic fibers of blood vessels and adjacent connective tissue, with no staining observed in cardiomyocytes themselves . High expression has also been documented in human ovary, lung, placenta, skin, spleen, and testis tissues . This distribution pattern aligns with MFAP4's proposed role in maintaining elastic fiber integrity and function.

What are the common applications for MFAP4 antibodies in research?

MFAP4 antibodies are utilized across multiple research applications, with various antibody products optimized for specific techniques. Based on the search results, the most common applications include:

The application range demonstrates MFAP4 antibodies' versatility in both qualitative and quantitative research approaches . When implementing these techniques, researchers should optimize antibody concentrations for their specific experimental conditions as dilutions may be sample-dependent .

What species reactivity is observed with commercially available MFAP4 antibodies?

Commercial MFAP4 antibodies demonstrate varying cross-reactivity profiles depending on the specific product. Based on the search results, most commonly available antibodies show reactivity against human MFAP4, with many also cross-reacting with mouse and rat orthologs . Some antibodies exhibit broader reactivity profiles spanning multiple species including human, mouse, rabbit, rat, bovine, dog, guinea pig, and horse . This cross-reactivity is particularly valuable for comparative studies across species models. When selecting an MFAP4 antibody for research, it's important to verify its reactivity against the target species of interest, as sequence conservation may vary across different regions of the protein .

How should MFAP4 antibodies be validated for experimental use?

Thorough validation of MFAP4 antibodies is essential for ensuring experimental reliability. A comprehensive validation approach should include:

• Specificity testing: Confirm target specificity through Western blot analysis, showing the expected band at approximately 36 kDa (observed) or 29 kDa (calculated) . Multiple tissue types with known MFAP4 expression patterns should be used as positive controls (e.g., human kidney, mouse heart, mouse lung, rat lung) .

• Cross-reactivity assessment: If working across species, validate the antibody using samples from each species of interest. Based on the search results, human, mouse, and rat samples have been successfully used for validation with certain antibodies .

• Application-specific validation: For each intended application (WB, IHC, IF, etc.), perform preliminary experiments to confirm appropriate signal-to-noise ratios and optimize experimental conditions.

• Negative controls: Include appropriate negative controls, such as isotype controls or tissues known to lack MFAP4 expression. Cardiomyocytes have been noted to show no MFAP4 staining and could serve as internal negative controls in cardiac tissue sections .

• Technical replicates: Perform experimental replicates to assess reproducibility, particularly important given the reported intra- and inter-assay variation in MFAP4 quantification (8.7% and 6.6%, respectively) .

What are the optimal protocols for detecting MFAP4 in different sample types?

Detection protocols for MFAP4 should be optimized based on the sample type and application:

For tissue samples (IHC):

• Antigen retrieval: Optimal results have been reported using TE buffer pH 9.0, although citrate buffer pH 6.0 may serve as an alternative .

• Antibody dilution: 1:20-1:200, optimized based on the specific antibody and tissue type .

• Detection system: Standard immunohistochemical detection methods are applicable, with particular attention to visualization in elastic fiber-rich regions.

For protein detection (Western Blot):

• Sample preparation: Tissue homogenization in appropriate lysis buffers that preserve protein integrity.

• Antibody dilution: 1:500-1:3000, depending on antibody sensitivity and protein abundance .

• Expected molecular weight: Look for bands at approximately 36 kDa, which represents the observed molecular weight of MFAP4 despite its calculated weight of 29 kDa .

For serum/plasma quantification (ELISA/AlphaLISA):

• The sandwich ELISA approach using two monoclonal anti-MFAP4 antibodies has been validated for measuring systemic MFAP4 .

• Practical working range: 4-75 U/ml for sandwich ELISA .

• Sample dilution: Final dilution of 1:100 has been reported for serum samples in AlphaLISA technique .

• Reference ranges: Average concentration in serum was found to be 18.9 ± 8.4 U/ml (95% CI: 18.5-19.4, median 17.3 U/ml) in a twin cohort study .

How do pre-analytical variables affect MFAP4 measurements in clinical samples?

Understanding pre-analytical variables is crucial for accurate MFAP4 quantification in clinical research:

According to the search results, sample handling and processing appear to influence MFAP4 measurements only marginally . This suggests that MFAP4 is relatively stable in clinical samples compared to many other biomarkers. Nevertheless, researchers should consider:

• Sample tube type: While specific data on tube types is not detailed in the search results, the validation study evaluated this variable and found minimal impact .

• Time and temperature conditions: The stability of MFAP4 under various storage conditions was assessed, with findings suggesting reasonable stability . Standard laboratory practices for protein preservation should still be followed.

• Freeze-thaw cycles: While not explicitly mentioned in the search results, repeated freeze-thaw cycles should generally be avoided to maintain protein integrity.

• Standardized collection protocols: To minimize variability, researchers should establish consistent sample collection, processing, and storage protocols.

The robustness of the MFAP4 assay to pre-analytical variables makes it particularly valuable for clinical studies where sample handling conditions may vary .

What biological and demographic factors influence basal MFAP4 variability?

Several demographic and biological factors have been identified that influence baseline MFAP4 levels:

A twin cohort study of 1,417 Danish twins (aged 18-67 years) identified multiple factors affecting basal systemic MFAP4 (sMFAP4) variability . The mean structure model demonstrated that:

• Waist-hip ratio: Body composition as measured by waist-hip ratio influences MFAP4 levels, with interactions observed with gender .

• Age: Aging appears to affect MFAP4 concentrations, also with gender-specific patterns .

• Cigarette smoking status: Smoking behavior impacts MFAP4 levels, with gender-specific interactions noted .

• Gender: Many of the above factors interact with gender, suggesting sex-specific regulation of MFAP4 .

• Genetic factors: Heritability analysis revealed a relatively low heritability (h² = 0.24) for MFAP4, indicating that genetic factors contribute approximately 24% to the observed variation, while environmental factors account for the majority .

Researchers conducting clinical studies involving MFAP4 should consider these variables in their study design and statistical analyses to account for these confounding factors.

What is the evidence supporting MFAP4 as a biomarker in fibrotic diseases?

MFAP4 has demonstrated promise as a biomarker in various fibrotic conditions:

Systemic MFAP4 (sMFAP4) is significantly elevated in samples from patients suffering from hepatic cirrhosis, suggesting its utility as a biomarker for liver fibrosis . This elevation likely reflects the increased extracellular matrix turnover characteristic of fibrotic diseases. The low heritability and relatively limited basal variation of sMFAP4 further support that increased levels primarily reflect disease-induced processes rather than genetic determinants .

MFAP4's localization to elastic fibers and connective tissue in the extracellular matrix provides a biological rationale for its potential as a biomarker of tissue remodeling . Its presence in various tissues suggests that MFAP4 may reflect ECM remodeling beyond liver fibrosis, potentially extending to other fibrotic diseases affecting elastic fiber-rich tissues such as the lungs and blood vessels.

The validated ELISA methodology for MFAP4 quantification provides a reliable technique for clinical biomarker studies, with acceptable intra- and inter-assay variation (8.7% and 6.6%, respectively) .

How can MFAP4 detection be optimized for clinical research studies?

For researchers designing clinical studies involving MFAP4 as a biomarker, several methodological considerations can optimize detection and interpretation:

• Assay selection: The validated sandwich ELISA based on two monoclonal anti-MFAP4 antibodies offers robust measurement capability with a practical working range of 4-75 U/ml . Alternatively, the AlphaLISA technique provides another validated approach for MFAP4 quantification in serum samples .

• Reference population: Researchers should establish appropriate reference ranges for their specific population. The reported average concentration of sMFAP4 in serum was 18.9 ± 8.4 U/ml in a Danish twin cohort, which may serve as a general reference .

• Confounding factors: Study designs should account for known confounders including waist-hip ratio, age, gender, and smoking status, either through exclusion criteria, stratification, or statistical adjustment .

• Sample timing: For longitudinal studies, consistent timing of sample collection is important to minimize variability, although MFAP4 appears relatively stable to pre-analytical handling .

• Complementary biomarkers: Combining MFAP4 with other established biomarkers of ECM remodeling may enhance diagnostic or prognostic value in fibrotic disease research.

The robustness of MFAP4 measurement methods and its disease-specific elevation pattern make it a promising candidate for clinical research into fibrotic conditions, particularly when methodological considerations are carefully addressed.

What are common technical challenges when working with MFAP4 antibodies?

Researchers working with MFAP4 antibodies may encounter several technical challenges:

• Molecular weight discrepancy: The observed molecular weight of MFAP4 (approximately 36 kDa) differs from its calculated molecular weight (29 kDa) . This discrepancy, likely due to post-translational modifications, could lead to confusion in Western blot analysis if researchers are strictly looking for a 29 kDa band.

• Antigen retrieval optimization: For IHC applications, antigen retrieval conditions may need optimization. While TE buffer at pH 9.0 is recommended, alternative approaches using citrate buffer at pH 6.0 may be necessary depending on sample fixation and tissue type .

• Cross-reactivity considerations: When studying MFAP4 across different species, antibody cross-reactivity should be carefully validated. Though several antibodies show reactivity with human, mouse, and rat MFAP4, specificity for other species may vary .

• Background signal in ECM-rich tissues: Given MFAP4's localization to elastic fibers and ECM, tissues rich in these components may present challenges in distinguishing specific from non-specific staining. Proper controls and optimization of blocking steps are essential.

• Variable expression levels: MFAP4 expression varies considerably across tissues, necessitating optimization of antibody dilutions for each tissue type. Tissues known to express high levels of MFAP4 (such as lung, ovary, skin) may require higher antibody dilutions than tissues with lower expression .

How can researchers optimize dilution and detection conditions for MFAP4 antibodies?

Optimization strategies for MFAP4 antibody applications include:

• Dilution optimization through titration:

  • For Western blot: Start with a dilution range of 1:500-1:3000 and optimize based on signal-to-noise ratio .

  • For IHC: Begin with a dilution range of 1:20-1:200, conducting a dilution series to determine optimal concentration .

• Application-specific conditions:

  • For IHC: Consider testing both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0) to determine which provides optimal staining with minimal background .

  • For Western blot: Test various blocking agents (BSA, milk, commercial blockers) to minimize background while preserving specific signal.

• Sample-dependent optimization:

  • As noted in the search results, optimal dilutions may be sample-dependent, requiring testing with each specific tissue or sample type .

  • Include positive control samples with known MFAP4 expression patterns, such as human kidney, mouse heart, mouse lung, or rat lung tissues .

• Detection system selection:

  • For low abundance situations, consider enhanced chemiluminescence or amplification steps.

  • For IHC, DAB (3,3'-diaminobenzidine) or AEC (3-amino-9-ethylcarbazole) systems may be selected based on the tissue type and counterstaining needs.

• Validation across applications:

  • When transitioning between applications (e.g., from WB to IHC), re-optimization is necessary as optimal dilutions vary significantly between techniques.

How should researchers interpret variations in MFAP4 levels in clinical samples?

Interpreting MFAP4 variations in clinical research requires consideration of several factors:

• Normal range context: The established normal range in serum is approximately 18.9 ± 8.4 U/ml (95% CI: 18.5-19.4, median 17.3 U/ml) based on a twin cohort study . Deviations should be interpreted relative to this range while considering population differences.

• Disease-specific elevations: Significant elevations have been documented in hepatic cirrhosis . The degree of elevation may correlate with disease severity, though specific thresholds must be established through targeted clinical studies.

• Contributing factors: When interpreting variations, consider the influence of waist-hip ratio, age, gender, and smoking status, which have been identified as factors affecting basal MFAP4 levels . Statistical adjustments for these variables may be necessary for accurate interpretation.

• Longitudinal changes: In monitoring studies, the relatively low intra- and inter-assay variation (8.7% and 6.6%, respectively) provides confidence that observed changes exceeding these technical variations likely represent true biological changes .

• Heritability context: The relatively low heritability (h² = 0.24) suggests that environmental factors and disease processes, rather than genetic predisposition, are the primary determinants of MFAP4 variation . This strengthens the case for MFAP4 as a dynamic biomarker of disease activity.

What are the key considerations when comparing MFAP4 data across different studies?

When comparing MFAP4 findings across different research studies, several methodological considerations are important:

• Assay methodology standardization: Confirm whether studies used comparable detection methods. The sandwich ELISA approach described in the search results and the AlphaLISA technique represent validated methodologies, but direct comparison of absolute values between different assay platforms may not be valid without cross-calibration.

• Units and reference standards: Verify that studies report MFAP4 levels in consistent units (e.g., U/ml) and used comparable reference standards. The use of recombinant MFAP4 standards should ideally be traceable to common reference materials .

• Population characteristics: Consider demographic differences between study populations, particularly regarding the factors known to influence MFAP4 levels (age, gender, smoking status, waist-hip ratio) . These differences may explain apparent discrepancies between studies.

• Pre-analytical handling: While MFAP4 measurements appear relatively robust to sample handling variations , significant differences in collection and storage protocols between studies could potentially influence results.

• Antibody specificity: Different studies may use antibodies recognizing different epitopes of MFAP4, potentially affecting the detection of specific isoforms or post-translationally modified variants.

• Clinical context interpretation: Similar elevations in MFAP4 may have different clinical implications depending on the disease context, comorbidities, and treatment status of the studied populations.