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-contai ning isoform of cellular fibronectin (EDAcFN) was established for scre ening of large series of blood samples and various body fluids of diff erent 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 s amples greatly depend on the ratio of sample dilution. The linearity o f the assay was improved with sample dilution and the optimal dilution was 1:5. Stored diluted samples retained their cFN content at +4 degr ees C, -20 degrees C and -70 degrees C for months in contrast to sampl es stored undiluted. With this direct EIA the detection limit was 0.05 mu g/ml and the linear portion of the standard curve could be extende d above 30 mu g/ml. Thus, the cFN concentration of blood samples could be measured reliably without inhibition also in samples with very hig h concentration of cFN. This is particularly important when measuring blood samples from cancer patients, since these samples may contain mo re than 20 mu g/ml EDAcFN. The assay was standardized for blood sample s but, due to the possibility of sample dilution, it also enabled reli able quantification of EDAcFN in various other body fluids. Undiluted some of the samples with non-neutral pH (urine, bile) or with high vis cosity (seminal plasma) interfered with the assay. In addition to Mood samples, the EDAcFN concentration was determined in samples of urine, bile, amniotic fluid, cervicovaginal secretions, seminal fluid, cereb rospinal fluid, bronchoalveolar ravage fluid, pleural fluid and saliva . Thereby, this modified method was shown to be applicable to various body fluids.