Measurement of fibronectin in human body fluids

Two facts must be taken into consideration when quantifying fibronectin in biological samples: the sensitivity of the assay method and the appropriate handling of samples. A three-antibody, non-competitive ELISA was used in the present study. This procedure offers a simple, inexpensive and very sensitive technique for evaluating fibronectin: reproducible standard curves in the range 5-100 micrograms/l, 3% variability in the assay, and a detection limit of 0.5 ng/well that allows accurate measurement of fibronectin in segmental bronchoalveolar lavage and in ascites liquid samples. Typical dilution ranged from 1/10000 for plasma to 1/100 for bronchoalveolar lavages. For long-term storage of samples two procedures are recommended. First, a rapid freezing with liquid nitrogen, storage at -20 degrees C and thawing once at 37 degrees C avoiding refreezing. Losses of immunoreactive fibronectin after 35 days storage were 15, 15 and 22% in plasma, bronchoalveolar lavages and ascites fluid, respectively. Alternatively, samples can be stored as ammonium sulphate precipitates. This allows the easy availability of the samples when assays have to be repeated, and avoids fractionation in vials and freeze-thawing cycles. Using this storage procedure, losses are, however, slightly higher (22, 25, 25%, respectively, after 35 days).