title: Enzyme-linked immunosorbent assay characterization of basal variation and heritability of systemic microfibrillar-associated protein 4.
abstract: 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.
authors: Cardiovascular 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.
authors: Susanne Gjørup
authors: SG
authors: Sækmose
authors: Anders
authors: A
authors: Schlosser
authors: René
authors: R
authors: Holst
authors: Sofie Lock
authors: SL
authors: Johansson
authors: Helle
authors: H
authors: Wulf-Johansson
authors: Ida
authors: I
authors: Tornøe
authors: Jørgen
authors: J
authors: Vestbo
authors: Kirsten Ohm
authors: KO
authors: Kyvik
authors: Torben
authors: T
authors: Barington
authors: Uffe
authors: U
authors: Holmskov
authors: Grith Lykke
authors: GL
authors: Sørensen
mesh_terms: D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D002352:Carrier Proteins / Q000097:blood* / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D016326:Extracellular Matrix Proteins / Q000097:blood* / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000276:immunology; D006801:Humans; D008103:Liver Cirrhosis / Q000097:blood; D008297:Male; D008875:Middle Aged; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012016:Reference Values; D015203:Reproducibility of Results; D013045:Species Specificity; D014427:Twins; D049629:Waist-Hip Ratio; D055815:Young Adult
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C400752:MFAP4 protein, human; D011993:Recombinant Fusion Proteins