Quantification of fibronectin in extracts of human aortae by an ELISA

Abstract

A procedure for extraction and quantification of fibronectin in human aortic tissue is described in this paper. Dried, defatted samples of human aortic tissue were subjected to sequential extraction with (i) 0.89% NaCl, 10 mmol/1 Tris/HCI, pH 7.4, (ii) 5 mg/ml heparin, 2 mol/1 urea and (iii) collagenase digestion. More than 75% of hexosamine-containing molecules were solubilized by this procedure. Immunoblotting of extracted proteins separated by SDS-PAGE showed that extracted fibronectin had a mobility in the same range as that of plasma fibronectin. Fibronectin ELISA performed on these extracts gave dilution curves parallel to the standard curve, the sensitivity was 2.7 μg/l. Recoveries of a fibronectin standard added to the NaCl, heparin/urea and collagenase solutions during extraction were 97%, 90% and 84% respectively. Normal aortic tissue from 31 patients was subjected to the sequential extraction scheme and fibronectin quantification in the various extracts demonstrated that 4.52+1.79 μg was dissolved in the NaCl extracts, 5.41±2.28 in the heparin/urea extract and 1.08±0.43 in the collagenase digest, respectively. (Values are expressed as μg fibronectin/10 mg dry, defatted tissue (mean±SD)). Our results indicate that the ELISA method can be applied for the measurement of fibronectin in extracts of human aortic tissue. This might be useful in the study of diseases where alterations in arterial fibronectin content may be expected.

Key Words:

• aorta
• ELISA
• fibronectin