One-Step Enzyme-Linked Immunosorbent Assay (ELISA) for Plasma Fatty Acid-Binding Protein

Abstract

SUMMARY. To allow a more rapid determination of heart-type fatty acid-binding protein (FABP) concentration in plasma a direct non-competitive (sandwich-type) ELISA was developed which uses high-affinity monoclonal antibodies to FABP. Total performance time of the one-step immunoassay is 45 min. The standard curve was linear between 0·2-6/lg/L, and the within-run and between-run coefficients of variations were below 6 and II %, respectively. The serum FABP concentration measured in 79 healthy individuals was 1·6 (0'8) [mean (SD), range 0,3-5'0] /lg/L. The assay can be used for rapid plasma or serum FABP measurement in the early diagnosis of acute myocardial infarction. Additional key phrases: heart-type fatty acid-binding protein; monoclonal antibodiesFatty acid-binding protein (FABP) is a recently introduced plasma marker of acute myocardial infarction (AMI) in man.' 4 The plasma kinetics of F ABP (15 kDa) closely resemble those of myoglobin (18 kDa) in that significantly elevated plasma concentrations are found within 3 h after AMI which generally return to normal values within 12 to 24h. 5 7 These features make FABP a useful biochemical marker especially for the early assessment or exclusion of AMI,5.6 and for the monitoring of a recurrent infarction." Since FABP released from the heart after AMI is quantitatively recovered in plasma, FABP can also be used to estimate infarct size."As with myoglobin, small quantities of (hearttype) FABP are also found in skeletal muscle, which are released into the circulation following injury." However, as the ratio of the tissue concentrations of myoglobin over FABP is different in heart (4-5) and skeletal muscles (2G--70), the assessment of this ratio in plasma allows the discrimination between myocardial and skeletal muscle injury."The clinical application of FABP to confirm or exclude a diagnosis of AMI soon after admission Correspondence: Jan F C Glatz PhD. requires a rapid test system, which is not yet available. Because FABP does not exhibit enzymatic activity, its plasma concentration has to be measured immunochemically. However, reported immunochemical assays for FABP take about 2 to 5 h to complete. 2,3,5,9 Here we describe a one-step sandwich-ELISA for FABP in plasma which uses high-affinity monoclonal antibodies, and shows a high sensitivity with a total performance time of only 45 min. MATERIALS AND METHODSCollection of blood samples For determination of the reference range of F ABP in serum and plasma, blood samples were withdrawn from 79 healthy subjects (61 men, median age 35, range 2G--51 years; and 18 women, median age 38, range 23-51 years). Subjects were taking no medication. Blood samples were obtained between 9 and 12 am and collected in glass tubes (preparation of serum). For IO subjects, a second blood sample was collected in glass tubes containing dry heparin (preparation of plasma). After centrifugation at 1500g for IOmin, serum or plasma was collected and stored at -70°C until analysis. 263

Weight Reduction and the Impaired Plasma-Derived Free Fatty Acid Oxidation in Type 2 Diabetic Subjects

“…The homogenates were subsequently sonicated four times for 15 s each time and centrifuged at 10,000 ϫ g for 2 min at 4 C to remove cell debris. The tissue content of heart-type or muscle-type FABPc in skeletal muscle was measured by means of a newly developed enzyme-linked immunosorbent assay (Hycult Biotechnology, Uden, The Netherlands), using recombinant human FABP as standard (24). Citrate synthase was determined according to the method of Shepherd and Garland (25), whereas 3-hydroxyacyl-coenzyme A dehydrogenase was assayed according to the method of Bergmeyer (26).…”

The effect of weight reduction on skeletal muscle UCP2 and UCP3 mRNA expression and UCP3 protein content in Type II diabetic subjects

“…Tissue content of heart-type or muscle-type cytoplasmatic fatty acid-binding protein (FABP) in skeletal muscle was measured by means of a newly developed ELISA, using recombinant human FABP as standard [30].…”

Fatty Acid- and Cholesterol Transporter Protein Expression along the Human Intestinal Tract

“…Since intestinal samples contained both enterocytes and myocytes, we also measured H-FABP as a marker of muscle content with a direct non-competitive sandwich-type ELISA using monoclonal antibodies obtained from Hycult biotechnology (HK 403; Uden, the Netherlands) as described previously [76]. These data showed significantly higher H-FABP in jejunum (1.7360.30) as compared to duodenum, ileum (1.0960.19), proximal colon (0.9760.16) and distal colon (0.9060.17), Because of this, we concluded that the biopsy muscle content was for an unknown reason higher in our jejunum biopsies and this might have resulted in an underestimation of cholesterol transport protein levels, which are only expressed in enterocytes.…”