Design and validation of a new immunoassay for soluble forms of thrombomodulin and studies on plasma.
Thrombomodulin (TM), purified from human placental homogeneate by affinity chromatography on DIP-Thrombin agarose, was used to develop monoclonal antibodies (MAbs). Two of them, 3E2 and 24FM (both IgG1, K), which were not calcium-dependent, were found convenient for developing a two-site enzyme immunoassay. Testing of recombinant and truncated forms of TM26 demonstrated that the species containing the amino terminus 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 interassay reproducibilities were, respectively, below 7.4% and 8.6%, whereas 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 individuals between the ages of 21 and 55 (28 males and 34 females). This new assay is a convenient tool for measuring plasma TM and establishing its diagnostic and predictive value in diseases associated to endothelial damage.
Amiral
J
J
Serbio Research Laboratory, Gennevilliers, France.
Adam
M
M
Mimilla
F
F
Larrivaz
I
I
Chambrette
B
B
Boffa
M C
MC
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008297:Male; D008875:Middle Aged; D018180:Thrombomodulin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D018180:Thrombomodulin