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Reagent and method for assaying thrombin-antithrombin complex

Inactive Publication Date: 2018-04-19
MITSUBISHI CHEM MEDIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention solves the problem of selecting the best combination of antibodies for use in immunological assay methods by efficiently selecting antibodies for latex agglutination assay that provide excellent fidelity, sensitivity, and specificity. The reagent provided in this invention can accurately measure a tiny amount of TAT in a biological sample while circumventing the effect of background substances and can be used on a general-purpose auto-analyzer. Additionally, the invention provides a reagent with high reactivity and specificity by keeping the pH during the reaction in the weak acidic range.

Problems solved by technology

However, the abundance of TAT complex is approximately 1 / 100,000 of the abundance of free antithrombin and, therefore, the measurement of TAT complex is not easy.
However, any of them are assay methods requiring separation between solid and liquid phases (B / F separation), which need laborious washing operations by hands or by special machines.
However, there is no report on any reagent that enables the precise concentration of TAT complexes in human samples to be measured using latex agglutination assay.
Patent Document 4 discloses an assay of TAT by a sandwich method using an antibody with no cross-reactivity but the assay lacks sufficient sensitivity for clinical use.
There has been no attempt so far to allow the reaction to proceed at an acidic pH in the TAT assay by latex immunoagglutination assay.

Method used

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  • Reagent and method for assaying thrombin-antithrombin complex
  • Reagent and method for assaying thrombin-antithrombin complex
  • Reagent and method for assaying thrombin-antithrombin complex

Examples

Experimental program
Comparison scheme
Effect test

example 1

on of Synthetic TAT Complexes

[0088]A commercially available human thrombin formulation (manufactured by Japan Blood Products Organization) and a commercially available antithrombin formulation (manufactured by Japan Blood Products Organization) were separately diluted with PBS (produced by dissolving Dulbecco's PBS (−) powder “Nissui (manufactured by Nissui Pharmaceutical Co., Ltd.)” to a concentration of 9.6 g / L) and those dilutions were mixed at a molar ratio of 1:3 and then allowed to react at 37° C. for 30 minutes. After the 30 minutes, DFP (diisopropyl fluorophosphate, manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 0.75 mM to stop the reaction.

[0089]Because unreacted thrombin and antithrombin were contained in the obtained reactant, purification was performed with a Hiload 26 / 60 Superdex 200 HR (manufactured by GE Healthcare) previously equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 500 mM NaCl.

[0090]TAT fractions were recovere...

example 2

on of an Anti-TAT Antibody

[0091]A cell fusion method was carried out according to the method described in Tamie Ando and Tatsuo Iwasaki, “Monoclonal Antibody / Hybridoma and ELISA” (Kodansha Ltd.).

[0092]The synthetic TAT complex prepared in Example 1 in an amount of 50 μg was mixed with Freund's complete adjuvant (manufactured by DIFCO) to provide an administered antigen.

[0093]The antigen was administered to BALB / c mice (female, four weeks old) three times at an interval of two weeks and 25 μg of the antigen, that is, half the amount of the original administered antigen was injected intravenously at the fourth administration.

[0094]One week later, lymphocytes were isolated from the spleen and mixed with P3x63-Ag.8 myeloma cells and then fused using polyethylene glycol (PEG 4000, manufactured by Merck).

[0095]Hybridoma cells were selected in HAT selection medium and then screened one week later for hybridoma clones producing an antibody of interest on the basis of the binding activity fo...

example 3

on of Anti-Thrombin Antibody

[0099]Anti-thrombin antibodies were obtained by a method similar to that in Example 2 using thrombin as an immunizing antigen. Antibodies specifically reacting with thrombin were selected and one of those clones was used as an anti-thrombin antibody (T-1).

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PUM

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Abstract

A thrombin-antithrombin complex (TAT) assay reagent is described in which the thrombin-antithrombin complex (TAT) assay reagent includes an antibody bound to a latex particle which binds to the antithrombin part of the complex, and an antibody bound to a latex particle which binds to the thrombin (T) part of the complex. The antibody binding to the antithrombin part of the complex has a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin and the assay reagent is preferably designed to achieve a pH of 5.8 to 6.6 during the assay.

Description

TECHNICAL FIELD[0001]The present invention relates to a reagent and a method for assaying the thrombin (T)-antithrombin (AT) complex (TAT) in a biological sample.BACKGROUND ART[0002]The thrombin-antithrombin complex (TAT) is a protein complex generated in blood in the course of blood coagulation and the quantification of TAT complexes in blood is useful for the diagnosis of thrombosis, such as disseminated intravascular coagulation (DIC). However, the abundance of TAT complex is approximately 1 / 100,000 of the abundance of free antithrombin and, therefore, the measurement of TAT complex is not easy.[0003]Examples of a currently prevailing TAT quantification method include reagent kits employing enzyme immunoassay (ELISA) technologies, such as Enzygnost® TAT micro from Siemens AG, and reagent kits employing chemiluminescent enzyme immunoassay (CLEIA) technologies, such as STACIA® CLEIA TAT from LSI Medience Co. However, any of them are assay methods requiring separation between solid ...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/53
CPCG01N33/54333G01N33/5306G01N33/54353G01N2333/974G01N33/573G01N33/6893G01N33/54313G01N2333/8128G01N2800/224G01N2800/226G01N33/86G01N33/545G01N33/543G01N33/6854G01N33/53
Inventor YOSHIDA, TATSUYAYANG, YUHANG
Owner MITSUBISHI CHEM MEDIENCE
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