R. Bos et al., A BIO-IMMUNO ASSAY TO DETERMINE FREE TISSUE-TYPE PLASMINOGEN-ACTIVATOR (T-PA) IN STABILYTE(R) PLASMA, Fibrinolysis, 8, 1994, pp. 163-165
This article describes a two-step bio-immuno assay (BIA), which determ
ines active (free) tissue-type plasminogen activator (t-PA) in (acidif
ied) plasma. Plasma samples, diluted in buffer of pH 6.0, are added to
the wells of a microtiter plate containing an immobilised monoclonal
antibody which binds t-PA without affecting t-PA activity. After an ov
ernight incubation at 4 degrees C, bound t-PA activity is determined b
y incubating the wells at 37 degrees C with plasminogen, CNBr-digested
fibrinogen and D-Val-Leu-Lys-pNA, followed by measurement(s) of the a
bsorbance (A) at 405 nm at timed intervals. The Delta A/t(2) or, alter
natively, the Delta A after 4 hours (endpoint determination) give an a
ccurate value for the active t-PA concentration. The assay has a lower
detection limit of 3 pg t-PA/ml and does not discriminate between var
ious forms of active t-PA. The intra-assay coefficients at 0.2 and 0.0
5 ng t-PA/ml were 5.5% and 4.3%, respectively. Active t-PA values in s
amples (N = 66) determined with this assay and with the t-PA BIA from
Chromogenix correlated relatively well (r = 0.78). Within a subset of
these samples (N = 18), a negative correlation with both tPA/PAI-1 (r
= 0.79) and PAI-1 antigen (r = 0.78) values was observed. Surprisingly
, no correlation was found with values for t-PA antigen (r = 0.21).