DESIGN AND VALIDATION OF A NEW IMMUNOASSAY FOR SOLUBLE FORMS OF THROMBOMODULIN AND STUDIES ON PLASMA

Thrombomodulin (TM), purified from human placental homogeneate by affi nity chromatography on DIP-Thrombin agarose, was used to develop monoc lonal antibodies (MAbs). Two of them, 3E2 and 24FM (both IgG(1), K), w hich were not calcium-dependent, were found convenient for developing a two-site enzyme immunoassay. Testing of recombinant and truncated fo rms of TM(26) demonstrated that the species containing the amino termi nus including the lectin-like domain and the epidermal growth factor ( EGF)-like domains 1-4 were fully measured. The working range was from 2 to 100 ng/ml with a detection threshold of 2 ng/ml. Intraassay and i nterassay reproducibilities were, respectively, below 7.4% and 8.6%, w hereas recovery of purified TM was between 88 and 114% in plasma. Mean plasma concentration was 42.1 (+/-11.3) ng/ml (males 51.8 +/- 7.9 ng/ ml, females 34.8 +/- 7.8 ng/ml) and it was established on 62 normal in dividuals between the ages of 21 and 55 (28 males and 34 females). Thi s new assay is a convenient tool for measuring plasma TM and establish ing its diagnostic and predictive value in diseases associated to endo thelial damage.