Establishment of Enzyme Immunoassay of Human Thrombomodulin in Plasma and Urine Using Monoclonal Antibodies

Abstract

SummaryThrombomodulin, TM, is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Soluble TM is present in plasma and urine of normal subjects. Enzyme immunoassay, EIA, for human TM was developed using mouse monoclonal antibodies against human placental TM in this paper. We obtained four types of the monoclonal antibodies against human placental TM. EIA sandwich method using three types of the monoclonal antibodies enabled us to measure almost all of 6 and 7 TM subspecies in plasma and urine, respectively, except 1 subspecies, 31 kDa TM. There was no interference from other components of plasma and urine in the assay conditions. Titration curves of purified TM in buffer or in normal plasma were linear within the range from 0.08 to 10 ng/ml. The coefficient of variation at 0.08 ng/ml TM was 4.7%. TM titer with buffer, assayed by this method, was reduced by the addition of thrombin at the final concentration of 20 U/ml, but the titer with plasma was not reduced even at 100 U/ml. These concentrations of thrombin are far larger than those which would be formed in circulation. TM levels in plasma and urine of normal subjects collected in the morning were 35.2 ± 8.32 ng/ml (n = 346) and 111 ± 31.6 ng/ml (n = 33), respectively. TM level in plasma did not differ from the level in serum. Circadian fluctuation of plasma TM was not significant in 10 normal adults, although a tendency of increase in TM excretion to urine was found rather in the day time than the other times. It was concluded that this EIA is reliable for TM assay in human plasma and urine, which will reflect activation or injury of endothelial cells.