In-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content

An in vitro detection and content technology, applied in measurement devices, preparation of test samples, instruments, etc., can solve the problems of expensive equipment, long detection time, low detection limit, etc., and achieve simple operation, low detection limit, diagnosis sensitive effect

Active Publication Date: 2011-06-22
LIAONING MEDI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a kind of in vitro detection method of TAFI content, and it has solved the defects such as expensive equipment that exists in the prior art, detection time is longer, compared with other detection methods, has simple and convenient operation, fast, low cost, diagnosis Sensitive, low detection limit and other advantages, as an auxiliary diagnosis for clinical prediction of cardiovascular and cerebrovascular diseases, can significantly improve the detection rate and accuracy of the disease, which is conducive to early detection and early treatment of the disease; reduce unnecessary Pain and medical expenses, improve the quality of life of the examinee

Method used

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  • In-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content
  • In-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content
  • In-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content

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preparation example Construction

[0046] 2. Preparation of coating antibody and enzyme-labeled antibody

[0047] Coating antibody and enzyme-labeled antibody are mouse anti-human monoclonal antibodies prepared against recombinant human TAFI antigen, and the mouse anti-human monoclonal antibodies include 3B1 and 4E7.

[0048] Hybridoma preparation: use purified human plasma TAFI as the immunogen, and immunize BALB / c mice according to conventional methods; take the splenocytes and fuse them with SP2 / 0 myeloma cells; screen the hybridoma cells, and positive wells are limited to 3 times Cloning by dilution method, and finally obtained two hybridoma cell lines that continuously and stably secrete anti-human TAFI monoclonal antibody, named 3B1 and 4E7 respectively;

[0049] Antibody identification: According to the instructions of Roche’s antibody subclass kit, the types of the two monoclonal antibodies were determined to be IgG1, κ; using simulated plasma as a control, Western blot and ELISA analysis showed that th...

Embodiment 1

[0078] Taking human plasma as an example, the method of the present invention is used to measure the TAFI content of 58 patients' plasma samples diagnosed as myocardial infarction and 30 healthy plasma samples. The specific operation steps are as follows:

[0079] Step 1 Collect the anticoagulated blood and immediately centrifuge it at 3000 rpm for 10 minutes at 4°C within 30 minutes, take the supernatant as the sample to be tested, and store it at -20°C.

[0080] Step 2: Use an in vitro detection kit to measure the TAFI content of the sample to be tested.

[0081] The contents of the in vitro detection kit used include: a microtiter plate coated with an antibody that captures the TAFI antigen of the sample to be tested and forms an antigen-antibody complex on the solid phase, a sample diluent for diluting the sample to be tested, and used for making calculations The TAFI standard of the standard curve of the TAFI antigen content in the sample to be tested is used to wash awa...

Embodiment 2

[0095] The method of the invention can also measure the TAFI content in human urine. Collect human morning urine, centrifuge at 1500 rpm for 5 minutes at 4°C within 30 minutes, and take the supernatant as the sample to be tested. Dilute the sample to be tested 100 times with the sample diluent. Subsequent steps are the same as in Embodiment 1.

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Abstract

The invention discloses an in-vitro assay method for thrombin-activatable fibrinolysis inhibitor (TAFI) content. The method is used for in-vitro assay of the TAFI content by using the biological characteristic that polyamidoamine (PAMAM) dendrimer can be combined with an antibody in combination with the conventional enzyme linked immunosorbent assay (ELISA) method to develop a new in-vitro diagnostic method for a TAFI, so that the flexibility of TAFI in-vitro assay is improved. In the method, a TAFI content in-vitro assay kit comprises an antibody-coated elisa plate, sample diluent, a TAFI standard product, cleaning solution, a PAMAM-labeled horseradish peroxidase bound antibody, antibody diluent, color development buffer solution, 30 percent hydrogen peroxide, o-phenylendiamine and stop solution. The defects of expensive equipment, longer assay time and the like existing in the prior art are overcome; compared with methods using other assay kits, the method has the advantages of low cost, sensitivity of diagnosis, low detection limit and the like, is easy and convenient to operate quickly and is used for auxiliary diagnosis, the detection rate and the accuracy of diseases are improved, and the aims of early detection and early treatment are fulfilled; and unnecessary pain and medical expenditure of an assayed person are reduced, and the quality of life of the assayed person is improved.

Description

technical field [0001] The invention relates to a new method for detecting the content of thrombin-activatable fibrinolysis inhibitor (TAFI) for clinical prediction of the possibility of occurrence of cardiovascular and cerebrovascular diseases, especially for detecting human individual biological In vitro detection method of TAFI content in samples such as blood, serum, plasma, urine, mucus, feces, cerebrospinal fluid, pleural effusion, ascites, saliva, tissue or cells, etc. In medical testing, it mainly uses the in vitro determination of TAFI content in human individual biological samples to assist in the diagnosis and treatment of related diseases such as cardiovascular and cerebrovascular diseases, acute promyelocytic leukemia, endogenous coagulation system defects, and inflammation. Large-scale clinical census. Background technique [0002] As we all know, thrombotic disease is a kind of disease that seriously affects health, especially cardiovascular and cerebrovascul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543G01N33/535G01N1/28
Inventor 李文欣
Owner LIAONING MEDI BIOTECH CO LTD
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