Evaluation of a bioimmunoassay for t-PA activity and its relation to PAI-1activity and antigen levels

A chromogenic bioimmunoassay method for determination of t-PA activities ha s been evaluated on plasmas from healthy individuals and from thromboemboli c patients. The assay consists of an initial binding and washing step, wher eby plasma t-PA is bound to a specific monoclonal t-PA antibody, which is c oated on to microplate wells, followed by a chromogenic plasmin generation step, using the chromogenic plasmin substrate S-2403. Through collection of blood at pH 6 and through maintaining this pH during the binding step, in vitro inhibition of t-PA by PAI-1 is prevented and the resulting t-PA activ ity reflects the condition in plasma at the time of blood sampling. Through parallel incubation of plasma at pH 8, an estimation of the PAI-I activity is obtained from the ratio of t-PA activities at pH 6 and 8. A median t-PA activity of 0.46 IU/mL (range 0.05-1.3, n = 69) was obtained for healthy individuals, whereas analysis of plasma from thromboembolic pat ients resulted in a median value of 0.31 IU/mL (range 0.04-1.5, n = 107). T hese results were in striking contrast to those obtained from 40 individual s from each group with parallel blood sampling in neutral citrate and incub ation at pH 8, where the corresponding activities were 0.03 IU/mL (range 0- 0.5) and 0.014 IU/mL (range 0-0.13) respectively, illustrating the severe l osses of t-PA activity obtained in vitro unless precautions to avoid inhibi tion by PAI-I are undertaken. Furthermore, the results showed an inverse correlation of the t-PA activity with PAI-I activity and antigen levels (r = 0.74-0.91). Elevated concentra tions of I-PA antigen in combination with elevated levels of PAI-I consiste ntly resulted in low t-PA activities. PAI-1 antigen concentrations above 70 ng/mL were always connected with t-PA activities below 0.2 IU/mL and with BIA t-PA ratios below 0.4. Prevention of t-PA inhibition by PAI-I at pH 8 t hrough addition of the monoclonal PAI-I antibody MAI-12 resulted in equal t -PA activities as obtained from incubation at pH 6. Altogether, the results suggest that the BIA t-PA assay performed an plasma samples collected in a cid medium provides a convenient fool for determination of basal steady sta te t-PA activities.