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Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay.
Anal Biochem. 1993 Feb 15; 209(1):70-8.AB

Abstract

Tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) coexist in numerous biological fluids, where their fibrinolytic activities are determined by the concentration of the free, uncomplexed species. A simple, sensitive method has been developed for the simultaneous determination of free t-PA and u-PA concentrations in biological fluids using a solid-phase immunoassay. Microtiter plates were coated with polyclonal goat antibodies and incubated with PA standards or unknown samples. The absorbed PAs were then assayed by incubation with a mixture of plasminogen, poly-L-lysine, and the chromogenic substrate H-D-norleucylhexahydrotyrosyllysine-p-nitroanilide. Free t-PA and free u-PA were detectable in human plasma and urine, and in conditioned media from different endothelial cell cultures. The method is sensitive, with lower limits of quantitation being 0.76 mU/ml (1.25 pg/ml) for free t-PA and 0.16 mU/ml (2.0 pg/ml) for free u-PA. There was no cross-reaction between the two PA species and the recovery in plasma was greater than 95% for both. The intra- and interassay coefficients of variation for t-PA and u-PA were 3.5-12.2 and 3.2-11.3%, and 2.4-11.8 and 1.6-10.4%, respectively. The presence of PA-inhibitor complexes and PA inhibitors in biological fluids did not interfere with the assay. Application of the assay has demonstrated that free u-PA levels are several fold higher than free t-PA levels in human plasma and in conditioned media of human vascular endothelial cells.(

ABSTRACT

TRUNCATED AT 250 WORDS)

Authors+Show Affiliations

Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8465964

Citation

Kuo, B S., and T D. Bjornsson. "Simultaneous Determination of Free Tissue-type and Free Urokinase-type Plasminogen Activators in Biological Fluids By a Solid-phase Immunoassay." Analytical Biochemistry, vol. 209, no. 1, 1993, pp. 70-8.
Kuo BS, Bjornsson TD. Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay. Anal Biochem. 1993;209(1):70-8.
Kuo, B. S., & Bjornsson, T. D. (1993). Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay. Analytical Biochemistry, 209(1), 70-8.
Kuo BS, Bjornsson TD. Simultaneous Determination of Free Tissue-type and Free Urokinase-type Plasminogen Activators in Biological Fluids By a Solid-phase Immunoassay. Anal Biochem. 1993 Feb 15;209(1):70-8. PubMed PMID: 8465964.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay. AU - Kuo,B S, AU - Bjornsson,T D, PY - 1993/2/15/pubmed PY - 1993/2/15/medline PY - 1993/2/15/entrez SP - 70 EP - 8 JF - Analytical biochemistry JO - Anal Biochem VL - 209 IS - 1 N2 - Tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) coexist in numerous biological fluids, where their fibrinolytic activities are determined by the concentration of the free, uncomplexed species. A simple, sensitive method has been developed for the simultaneous determination of free t-PA and u-PA concentrations in biological fluids using a solid-phase immunoassay. Microtiter plates were coated with polyclonal goat antibodies and incubated with PA standards or unknown samples. The absorbed PAs were then assayed by incubation with a mixture of plasminogen, poly-L-lysine, and the chromogenic substrate H-D-norleucylhexahydrotyrosyllysine-p-nitroanilide. Free t-PA and free u-PA were detectable in human plasma and urine, and in conditioned media from different endothelial cell cultures. The method is sensitive, with lower limits of quantitation being 0.76 mU/ml (1.25 pg/ml) for free t-PA and 0.16 mU/ml (2.0 pg/ml) for free u-PA. There was no cross-reaction between the two PA species and the recovery in plasma was greater than 95% for both. The intra- and interassay coefficients of variation for t-PA and u-PA were 3.5-12.2 and 3.2-11.3%, and 2.4-11.8 and 1.6-10.4%, respectively. The presence of PA-inhibitor complexes and PA inhibitors in biological fluids did not interfere with the assay. Application of the assay has demonstrated that free u-PA levels are several fold higher than free t-PA levels in human plasma and in conditioned media of human vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/8465964/Simultaneous_determination_of_free_tissue_type_and_free_urokinase_type_plasminogen_activators_in_biological_fluids_by_a_solid_phase_immunoassay_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(83)71084-5 DB - PRIME DP - Unbound Medicine ER -