Tissue-type plasminogen activator in plasma from breast cancer patients determined by enzyme-linked immunosorbent assay

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.

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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 ◽

10.1182/blood.v69.1.284.284 ◽

1987 ◽

Vol 69 (1) ◽

pp. 284-289 ◽

Cited By ~ 24

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.

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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 ◽

10.1182/blood.v69.1.284.bloodjournal691284 ◽

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.

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