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.

Characterization of Two Monoclonal Antibodies Against Human Tissue-Type Plasminogen Activator

1985 ◽  
Vol 53 (02) ◽  
pp. 170-175 ◽  
Author(s):  
Raymond R Schleef ◽  
Manjula Sinha ◽  
David J Loskutoff
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Association Constant ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Standard Procedures ◽  
Type Plasminogen Activator

SummaryA series of hybridoma clones, each producing monoclonal antibodies to human tissue-type plasminogen activator (t-PA), were prepared from mice by standard procedures. Two of these clones were selected for further study. One HI72A1, produced antibodies that bound to t-PA and strongly inhibited its activity, whereas another, LI72D1, produced antibodies that bound to t-PA but did not affect its activity. The specificity of these antibodies was assessed in immunoabsorption experiments. Both immunoprecipitated 125I-labeled t-PA, and both were specific since only t-PA was recognized in conditioned media collected from Bowes melanoma cells cultured in the presence of 3H-leucine. Neither antibody recognized urokinase. t-PA was desorbed from antibody HI72A1-Sepharose columns with 0.5 M NaCl, consistent with its relatively low association constant (Ka = 9.37 × 107 M-1). In contrast, a strong chaotropic agent (i.e., 2 M KI) was required to elute t-PA from antibody LI72D1 columns (Ka = 2.08 × 109 M-1). This latter high affinity antibody was employed to develop an immunoradiometric assay for t-PA having a sensitivity of 0.5 ng/ml.

Characterization of a Panel of Monoclonal Antibodies Against Human Tissue-Type Plasminogen Activator

Hybridoma ◽  
1988 ◽  
Vol 7 (2) ◽  
pp. 177-184 ◽  
Author(s):  
THOMAS M. REILLY ◽  
SANDRA K. FLINT ◽  
BERNIE G. McHUGH ◽  
KATHLEEN M. WILSBACH-VOLCHECK ◽  
PIETER B.M.W.M. TIMMERMANS
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

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.

Characterization of Human Tissue-Type Plasminogen Activator with Monoclonal Antibodies: Mapping of Epitopes and Binding Sites for Fibrin and Lysine

1992 ◽  
Vol 67 (01) ◽  
pp. 088-094 ◽  
Author(s):  
Ute Zacharias ◽  
Bernhard Fischer ◽  
Franz Noll ◽  
Horst Will
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Tissue Type ◽  
Reduced Form ◽  
Amidolytic Activity ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Catalytically Active ◽  
A Chain

SummaryThe study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding.Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D’). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA.The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F. Weak inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic activity is suppressed only by B-chain specific antibody 22.

Measurement of Urokinase-Type Plasminogen Activator (u-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies

1986 ◽  
Vol 56 (03) ◽  
pp. 411-414 ◽  
Author(s):  
V Darras ◽  
M Thienpont ◽  
D C Stump ◽  
D Collen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Microtiter Plates ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Murine Monoclonal Antibodies

SummaryAn enzyme-linked immunosorbent assay (ELISA) for the measurement of urokinase-type plasminogen activator (u-PA) was developed. Three murine monoclonal antibodies to single chain urokinase-type plasminogen activator (scu-PA) were isolated and shown to react with non-overlapping epitopes in scu-PA. Two of the three antibodies were coated onto microtiter plates and bound u-PA was quantitated with the third antibody conjugated to horseradish peroxidase. The assay was equally sensitive to Mr 54,000 u-PA in the single chain or two-chain form but did not respond to Mr 33,000 urokinase. The lower limit of sensitivity of the assay was 0.1 ng/ml in buffer and 1 ng/ml in plasma. Coefficients of variation of the assay at physiological levels of u-PA were 6.5 percent within assays and 13 percent between assays. The level of u-PA in normal resting plasma was 1.9 ± 0.66 ng/ml (mean ± SD, n = 54). The assay can be performed within one working day and provides an efficient, reproducible, and stable means for the measurement of u-PA in biological fluids. As such it may facilitate physiological and pharmacological studies of urokinase-type plasminogen activators in man.

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