Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-06-03
1994-10-04
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 13, 435962, 436527, 436531, 436548, 436 69, 53038825, 5303893, G01N 3353
Patent
active
053525833
DESCRIPTION:
BRIEF SUMMARY
Detailed Description of the Invention
a. Industrial Applicable Field
This invention relates to the immunological assay of the human tissue plasminogen activator-human plasminogen activator inhibitor complex in a human specimen. More detailedly, the invention relates to an immunological assay method for assaying with a high sensitivity and stably the amount of the complex in a human specimen, and a kit therefor.
The symbols used in the present description have the following meanings unless otherwise defined. activator inhibitor complex
b. Prior Art
Plasmin, an enzyme to dissolve fibrin, is formed from the conversion of plasminogen with a tissue plasminogen activator (tPA). In recent years, it was found that an inhibitor against this human tissue plasminogen activator [human plasminogen activator inhibitor (PAI)] exists in blood vessel endothelial cells, cutaneous cells, platelets, the placenta, etc., and forms without delay a complex with the human tissue plasminogen activator, and thereby inhibits a human tissue plasminogen activator activity (M. Philips et al, Biochem Biophys, Acta, 802, 99-110, 1984, S. Thorsen, Biochem Biophys Acta, 802, 111-118, 1984, T. Wun et al, J, Biol Chem. 262, 3646-3653, 1987, M. A. Sanzo et al, Biochem, 26, 7443-7449, 1987, Y. Sakata et al, J. Biol Chem. 263, 1960-1969, 1988). Further, there has been a clarification of the relationship between the activity value of the plasminogen activator inhibitor (PAI) in the blood and a disease (B. Wiman et al. Scand. J. Clin Lab Invest 45, 43-43, 1985, P. Vague et al, Metabolism 35, 250-253, 1986, A. Hamsten, Lancet 8549,3-8, 1987), and it is suggested that the plasminogen activator inhibitor (PAI) is an important control factor of the initiation mechanism of the blood coagulation fibrinogenolysis system. Therefore, when it is possible to know the concentration of PAI, tPA and the tPA-PAI complex in the blood, there is a large possibility that the abnormality and pathema of the fibrinogenolysis system can be monitored.
Presently, as assay methods for tPA there are methods by sandwich enzyme immunoassay (Japanese Laid-Open Patent Publication No. 174759/1984), kits on the market (Biopool Co., IMULYSE t-PA), etc., and on the other hand as methods for assay PAI there are methods using a radioactive substance (R. R. Schlef et al, J. Lab Clin Med 106, 408, 1985), a sandwich enzyme immunoassay kit using a monoclonal antibody (Biopool Co., IMULYSE PAI-I), etc.
Further, it is said that a very small amount of a tPA-PAI complex exists in the plasma, and as an assay method for this tPA-PAI complex there is an enzyme immunoassay method based on the fluorescence method, wherein a monoclonal antibody against PAI is used as an immobilized antibody, a polyclonal antibody against tPA is used as a labeled antibody and its fragment is labeled with .beta.-galactosidase.
This system to assay the tPA-PAI complex comprises reacting, first, the immobilized monoclonal anti-PAI-1 antibody with a specimen at 30.degree. C. for 3 hours, washing, reacting with the Fab' fragment of the polyclonal anti-tPA labeled with .beta.-galactosidase at 4.degree. C. overnight to complete the immunological logical reaction, decomposing 4-methyl-umbelliferyl galactoside, and assaying the formed 4-methylumbelliferone by the fluorescence method to quantitatively determine to tPA-PAI complex. Such method has problems that the reaction temperatures differ between the two immunological reactions, reaction time is necessary to be overnight and very long, and further a fluorescence photometer, a special measurement apparatus, is used, and thus it has been difficult to make the assay effectively in industrial and clinical aspects.
On the other hand, Amiral et al. propose, as an assay method for a tPA-PAI complex, a method for assaying the tPA-PAI complex using a monoclonal antibody against PAI and a monoclonal antibody against tPA (Thrombosis Research, Supplement VIII; 99-113, 1988). In this method two kinds of monoclonal antibodies are used and the tPA-PAI complex can be assayed with a rel
REFERENCES:
patent: 4148869 (1979-04-01), Deaton
R. Vogt et al., "Quantitative differences among various proteins as blocking agents for ELISA microtiter plates", in Journal of Immunological Methods, vol. 101, pp. 43-50 (1987).
Y. Takada et al., "Measurements of the Concentration of Free-Plasminogen Activator, Inhibitor (PAI-1) and Its Complexes with Tissue Plasminogen Activator", Chemical Abstract 109(25):225318.
Takada et al. Thrombosis Research, vol. 55, No. 5; 601-610 "Plasma levels of t-P A, free PAI-1 and a complex of t-PA with PAI-1 in human males and females at various ages" (1989).
J. Amiral et al., Thrombosis Research, Supplement VIII; 99-113, "Measurement of tPA and tPA---PAI-1 complexes by Elisa, using monoclonal antibodies: clinical relevance" (1988).
Hasegawa Ryoichi
Hino Shuichiro
Itoh Kazuhiko
Murakami Toshinobu
Noro Atsushi
Spiegel Carol A.
Teijin Limited
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