An improved method for quantification of extra domain A-containing cellular fibronectin (EDAcFN) in different body fluids.

A quantitative direct enzyme immunoassay for the extra domain A-containing isoform of cellular fibronectin (EDAcFN) was established for screening of large series of blood samples and various body fluids of different pH and viscosity. The method is based on the monoclonal antibody DH1 recognizing the extra domain A in cellular fibronectin (EDAcFN). Studies on the effect of dilution of plasma and serum samples in this direct assay indicated that the measured concentration of cFN in the samples greatly depend on the ratio of sample dilution. The linearity of the assay was improved with sample dilution and the optimal dilution was 1:5. Stored diluted samples retained their cFN content at +4 degrees C, and -20 degrees C and -70 degrees C for months in contrast to samples stored undiluted. With this direct EIA the detection limit was 0.05 micrograms/ml and the linear portion of the standard curve could be extended above 30 micrograms/ml. Thus, the cFN concentration of blood samples could be measured reliably without inhibition also in samples with very high concentration of cFN. This is particularly important when measuring blood samples from cancer patients, since these samples may contain more than 20 micrograms/ml EDAcFN. The assay was standardized for blood samples but, due to the possibility of sample dilution, it also enabled reliable quantification of EDAcFN in various other body fluids. Undiluted some of the samples with non-neutral pH (urine, bile) or with high viscosity (seminal plasma) interfered with the assay. In addition to blood samples, the EDAcFN concentration was determined in samples of urine, bile, amniotic fluid, cervicovaginal secretions, seminal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, pleural fluid and saliva. Thereby, this modified method was shown to be applicable to various body fluids.