Development and evaluation of a new kinetic assay for the quantitation of fibronectin gelatin-binding activity

A new rapid and sensitive kinetic assay that measures the gelatin-binding activity of fibronectin has been developed. This assay is based on the rate of fibronectin-mediated aggregation of covalently coupled latex-gelatin particles. The addition of human plasma and serum resulted in aggregation rates proportional to the concentration of fibronectin in the test sample. This assay was inhibited by the addition of gelatin, demonstrating substrate specificity. This new assay requires 50 microliter of sample and can be performed within 5 minutes. Particle aggregation rates were affected by pH, heparin, and coupled gelatin concentration per milligram of latex. Maximum aggregation rates were observed at pH 8.0. Heparin was not an absolute requirement for particle aggregation but enhanced rates up to 1 U heparin/ml with little additive effect at greater concentrations. Heparin had a relatively greater effect on assays performed in acidic buffers. The concentration of gelatin per milligram of latex was rate limiting up to approximately 50 micrograms gelatin/mg latex with little change in aggregation rates at higher concentrations. Good correlation between total antigenic fibronectin (electroimmunoassay) and fibronectin gelatin-binding activity (latex-gelatin kinetic aggregation assay) was demonstrated in plasma from normal blood donors. This new assay will allow further definition of the relationship between fibronectin gelatin-binding activity and antigenic fibronectin in normal and pathophysiologic states.