scholarly journals Measurement of free, one-chain tissue-type plasminogen activator in human plasma with an enzyme-linked immunosorbent assay based on an active site-specific murine monoclonal antibody

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 284-289 ◽  
Author(s):  
P Holvoet ◽  
J Boes ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Active Site ◽  
Venous Occlusion ◽  
Murine Monoclonal Antibody ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Abstract An enzyme-linked immunosorbent assay for free tissue-type plasminogen activator (t-PA) in human blood was developed based on a murine monoclonal antibody directed against the active site of t-PA. The lower limit of sensitivity of the assay applied to plasma is 2 ng/mL for one- chain t-PA but only 100 ng/mL for two-chain t-PA. Free t-PA in plasma taken at rest was found in 6 of 21 healthy subjects (4.5 +/- 0.8 ng/mL, mean +/- SD) and increased to 14 +/- 7.0 ng/mL after venous occlusion in 18 of these individuals. A linear correlation between total t-PA and free t-PA was observed with r = 0.92 (n = 18) and a slope of 1.08, indicating that t-PA released from the vessel wall circulates in the blood as the one-chain form. In 16 of 18 patients with deep vein thrombosis, the increase of total t-PA antigen after venous occlusion was comparable to that observed in controls, but the free t-PA was significantly lower or undetectable. The present assay for free t-PA may be useful for the investigation of the release and inhibition of t- PA under physiological, pharmacological, or pathological conditions in humans.

scholarly journals Measurement of free, one-chain tissue-type plasminogen activator in human plasma with an enzyme-linked immunosorbent assay based on an active site-specific murine monoclonal antibody

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 284-289
Author(s):  
P Holvoet ◽  
J Boes ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Active Site ◽  
Venous Occlusion ◽  
Murine Monoclonal Antibody ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

An enzyme-linked immunosorbent assay for free tissue-type plasminogen activator (t-PA) in human blood was developed based on a murine monoclonal antibody directed against the active site of t-PA. The lower limit of sensitivity of the assay applied to plasma is 2 ng/mL for one- chain t-PA but only 100 ng/mL for two-chain t-PA. Free t-PA in plasma taken at rest was found in 6 of 21 healthy subjects (4.5 +/- 0.8 ng/mL, mean +/- SD) and increased to 14 +/- 7.0 ng/mL after venous occlusion in 18 of these individuals. A linear correlation between total t-PA and free t-PA was observed with r = 0.92 (n = 18) and a slope of 1.08, indicating that t-PA released from the vessel wall circulates in the blood as the one-chain form. In 16 of 18 patients with deep vein thrombosis, the increase of total t-PA antigen after venous occlusion was comparable to that observed in controls, but the free t-PA was significantly lower or undetectable. The present assay for free t-PA may be useful for the investigation of the release and inhibition of t- PA under physiological, pharmacological, or pathological conditions in humans.

Assay of Human Tissue-Type Plasminogen Activator (t-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies to t-PA

1985 ◽  
Vol 54 (03) ◽  
pp. 684-687 ◽  
Author(s):  
Paul Holvoet ◽  
Hugo Cleemput ◽  
Désiré Collen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Human Tissue ◽  
Biological Fluids ◽  
Human Subjects ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryAn enzyme-linked immunosorbent assay (ELISA) for the measurement of human tissue-type plasminogen activator (t-PA) was developed. Microtiter plates were coated with a mixture of two monoclonal antibodies and bound t-PA was quantitated with a third monoclonal antibody linked to peroxidase. The lower limit of sensitivity of the assay was 0.2 ng of t-PA per ml. The concentration of antigen in citrated plasma of human subjects was found to be 3.4 ± 0.8 ng/ml. The assay had a good reproducibility with values of 3.8, 6.5 and 4.9 percent respectively for the intra-, inter-assay and inter-dilution variation coefficients. The results of the ELISA assay on plasma samples from patients during thrombolytic therapy with t-PA correlated very well, over a wide concentration range, with those obtained with a previously described two-site immuno-radiometric assay (r = 0.96). This ELISA with monoclonal antibodies constitutes a stable and reproducible set of reagents for the measurement of t-PA antigen in biological fluids, avoiding the disadvantages of the use of radioisotopes and of polyclonal antibodies.

Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood

1983 ◽  
Vol 50 (02) ◽  
pp. 518-523 ◽  
Author(s):  
C Kluft ◽  
A F H Jie ◽  
R A Allen
Keyword(s):  
Plasminogen Activator ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Functional Assay ◽  
Uterine Tissue ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Baseline Values

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

scholarly journals Structural Mapping of the Active Site Specificity Determinants of Human Tissue-type Plasminogen Activator

1997 ◽  
Vol 272 (35) ◽  
pp. 21713-21719 ◽  
Author(s):  
Martin Renatus ◽  
Wolfram Bode ◽  
Robert Huber ◽  
Jörg Stürzebecher ◽  
Dagmar Prasa ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Tissue Type ◽  
Site Specificity ◽  
Structural Mapping ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Specificity Determinants

scholarly journals An active-site titrant for human tissue-type plasminogen activator

1986 ◽  
Vol 239 (2) ◽  
pp. 477-479 ◽  
Author(s):  
R A Smith
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Rapid Release ◽  
Type Plasminogen Activator ◽  
Inverse Substrate

The reaction of recombinant tissue-type plasminogen activator with the inverse substrate 4-amidino-2-nitrophenyl 4′-anisate results in the rapid release of the chromogen 4-amidino-2-nitrophenol and the accumulation of the relatively stable 4-anisoyl-enzyme. Spectrophotometric monitoring of the reaction enables the operational molarity of the enzyme to be determined.

scholarly journals Blood cells participate in the fibrinolytic response to tissue-type plasminogen activator

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 564-567 ◽  
Author(s):  
MW Hammouda ◽  
LA Moroz
Keyword(s):  
Plasminogen Activator ◽  
Whole Blood ◽  
Cellular Response ◽  
Solid Phase ◽  
Venous Blood ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Phase Activity

Abstract Exercise and venous occlusion stimulate fibrinolysis. In addition to increased concentrations of tissue-type plasminogen activator (tPA) and increased plasmin-mediated fibrinolysis in plasma, these stimuli produce additional cellular-phase activity in blood that is of the same magnitude as the plasma response. To determine whether tPA alone, rather than other consequences of these stimuli, is responsible for the cellular response, the in vitro effects of tPA on whole blood, plasma, and calculated cellular-phase (whole blood minus plasma) activities were determined by solid-phase radiofibrin assay on venous blood from ten normal subjects (seven men, three women). At tPA concentrations encompassing physiological and therapeutic levels (5 to 100 ng/mL; 0.7 to 14 IU/mL), increments in whole blood were consistently in excess of those in companion plasma and represented increased cellular-phase activity equivalent in magnitude to the well-known increase in plasma activity. Fibrinolytic activity produced by 10 to 20 ng tPA/mL (1.4 to 2.8 IU/mL) was consistently detected in whole blood and plasma by 60 minutes, with higher concentrations (100 ng or 14 IU tPA/mL) detectable after a five-minute assay in all subjects. Thus, tPA alone, without invoking fibrinolytic factors extraneous to blood or other effects of exercise or venous occlusion, accounts for both cellular and plasma responses to these stimuli. The considerable cellular response, the mechanism of which remains to be elucidated, may constitute a determinant of individual therapeutic responsiveness to tPA.

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