An enzyme-linked immunoassay for direct measurement of the gelatin-binding capacity of human plasma fibronectin.
A new solid-phase enzyme immunoassay measuring the gelatin-binding capacity of plasma fibronectin has been developed. This assay is based on the direct and high-affinity interaction between fibronectin and gelatin coated to polyvinyl chloride plates. The amount of fibronectin bound to gelatin is then measured by sequential incubation with a specific rabbit anti-human fibronectin antiserum, with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies and with substrate. The final degradation of the substrate is read at 492-650 nm in an ELISA processor. The assay allows the accurate detection of fibronectin concentrations ranging from 1 to 20 micrograms/ml, is inhibited by the addition of gelatin to plasma, is highly reproducible (interplate CV less than 10%), requires 100 microliter of plasma only and has been fully automated. Significant linear correlations were noted between total antigenic fibronectin (measured by laser nephelometry) and fibronectin gelatin-binding capacity in plasma from 310 blood donors. Both parameters were higher in men than in women and significantly increased according to age. Dissociation between immunoreactive fibronectin and fibronectin gelatin-binding capacity was observed in two polytraumatized patients. This enzyme immunoassay therefore provides a new method to investigate functional alterations of the gelatin-binding domain of fibronectin in various pathological conditions.
D000328:Adult; D000367:Age Factors; D000368:Aged; D001322:Autoanalysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005353:Fibronectins / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding; D012737:Sex Factors; D014947:Wounds and Injuries / Q000097:blood
D005353:Fibronectins; D005780:Gelatin