Enzyme-linked immunosorbent assay characterization of basal variation and heritability of systemic
Susanne Gjørup Sækmose1, Anders Schlosser, René Holst
1Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark ; Department of Clinical Immunology, Region Sjaelland, Naestved Hospital, Naestved, Denmark ; Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
Background:
Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation.
Methods:
The sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years) by mixed-effect linear regression modeling.
Results:
The practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD) U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml). The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h(2) = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors.
Conclusions:
The described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively limited basal variation suggest that increased sMFAP4 reflects disease-induced processes.
Insights
This study validates a robust ELISA for measuring systemic microfibrillar-associated protein 4 (sMFAP4), a marker elevated in liver cirrhosis. Basal sMFAP4 levels show low heritability, suggesting disease influences these levels.
Area of Science:
- Biochemistry
- Biomarker Discovery
- Genetics
Background:
- Microfibrillar-associated protein 4 (MFAP4) is elevated in hepatic cirrhosis patients.
- Systemic MFAP4 (sMFAP4) variation may indicate diseases with increased extracellular matrix (ECM) turnover.
- MFAP4 is localized to connective tissue fibers in the ECM.
Purpose of the Study:
- Validate a robust enzyme-linked immunosorbent assay (ELISA) for sMFAP4 measurement.
- Identify confounders affecting basal sMFAP4 variability.
- Determine the genetic contribution to basal sMFAP4 variation.
Main Methods:
- A sandwich ELISA using monoclonal anti-MFAP4 antibodies was developed and optimized.
- Pre-analytical sample handling (tube type, time, temperature) was assessed.
- A Danish twin cohort (n=1,417) was analyzed using mixed-effect linear regression to determine variance structure and heritability.
Main Results:
- The ELISA demonstrated a working range of 4-75 U/ml with low intra-assay (8.7%) and inter-assay (6.6%) variation.
- Sample handling had minimal impact on MFAP4 measurements.
- Basal sMFAP4 levels (mean 18.9 U/ml) were influenced by waist-hip ratio, age, and smoking status interacting with gender.
- Heritability (h²) was low at 0.24.
Conclusions:
- The validated ELISA provides robust sMFAP4 measurements, useful for liver fibrosis assessment.
- Limited basal variation and low heritability suggest elevated sMFAP4 primarily reflects disease-specific processes.
- sMFAP4 serves as a valuable systemic biomarker for ECM-related diseases.
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