Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method

An antithrombin and thrombin technology, applied in the field of immunoassay, to achieve the effects of wide linear range, improved work efficiency and high detection sensitivity

Pending Publication Date: 2020-04-14
江苏三联生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no mature method of electr

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  • Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method
  • Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method
  • Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 detects the preparation method of the electrochemiluminescence kit of TAT

[0022] 1) Preparation of streptavidin-coupled magnetic particle working solution

[0023] The streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.5 mg / mL with a pH of 6.8 and a concentration of 0.1 mol / L PBS buffer containing 0.5 wt% bovine serum albumin, 0.05 wt% Triton X-100, 0.05 wt% proclin 300 and 0.05 wt% sodium azide;

[0024] 2) Preparation of biotinylated thrombin antibody

[0025] Take 1 mg of thrombin mouse monoclonal antibody, add appropriate amount of PBS buffer (0.05M, pH7.0-7.6) to adjust the total antibody concentration to 1 mg / ml, and put it into a dialysis bag for dialysis, changing it every 3-4 hours Dialysate, changed 3-4 times, after dialysis, transfer the antibody to a centrifuge tube or cryotube;

[0026] Accurately weigh 1 mg of biotin activated by N-hydroxysuccinim...

Embodiment 2

[0041] Example 2 The method for detecting thrombin using the kit of the present invention uses a fully automatic electrochemiluminescence immunoassay analyzer as a detection instrument, and loads the kit on the instrument for detection, the steps are as follows:

[0042] Add the sample (or calibrator or quality control), biotin-labeled thrombin antibody working solution and ruthenium-tripyridyl-labeled anti-anti-thrombin antibody working solution into the cuvette, and incubate at 37°C for 10 minutes to form the antibody- Antigen-antibody sandwich complex solution;

[0043] Add streptavidin-coated magnetic particle working solution to the antibody-antigen-antibody sandwich complex solution, and incubate at 37°C for 10 minutes to form a magnetic complex suspension;

[0044] placing the suspension of the magnetic complex in a magnetic field for magnetic separation, and flowing a cleaning solution to wash the magnetic complex;

[0045] The electrochemiluminescence substrate solut...

Embodiment 3

[0047] Embodiment 3 The research of reagent sensitivity

[0048] Reagent sensitivity is determined according to the lowest detection limit (LOB), which is carried out according to the following experimental method. Detect the zero-concentration calibrator 20 times, get the signal value (RLU) of 20 measurement results, calculate the mean M and standard deviation SD, and obtain the RLU value corresponding to M+2SD, according to the zero-concentration calibrator and the adjacent concentration The concentration-RLU value between the calibrator (adjacent concentration is 1ng / mL) is obtained by two-point regression fitting to obtain a linear equation, and the RLU value corresponding to M+2SD is brought into the above equation to calculate the corresponding Concentration, the lower limit of detection (LOB).

[0049] The measurement results in Table 1 below show that the detection limit (LOB) of the reagent by the above method is 0.017 ng / mL, and according to this result, it is deter...

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Abstract

The invention relates to an electrochemiluminescence kit for detecting a novel thrombus marker TAT (thrombin-antithrombin complex) in blood and a preparation method of the electrochemiluminescence kit. The prepared kit comprises a streptavidin coupled magnetic particle working solution, a biotin labeled thrombin antibody working solution, a terpyridyl ruthenium labeled antithrombin antibody working solution, a TAT calibrator and/or quality control substance working solution, a tripropylamine-containing electrochemiluminescence substrate solution and a cleaning solution. A luminescence system of the kit is electrochemiluminescence, a streptavidin-biotin signal amplification system is utilized, the detection sensitivity is high, the linear range is wide, the detection result repeatability ishigh, and accurate quantification of the thrombin-antithrombin compound can be achieved.

Description

technical field [0001] The invention relates to an electrochemiluminescence reagent kit and usage for detecting TAT (thrombin-antithrombin complex), belonging to the technical field of immune analysis. Background technique [0002] Thrombin is a multifunctional serine proteolytic enzyme with a sequence and structure similar to chymotrypsin. It contains two polypeptide chains, A and B, connected by an interchain disulfide bond. The β chain is a functional chain with a typical fold structure of serine proteolytic enzymes, which contains an active center located between two β sheet barrels, 2 external binding sites, 1 Na ion binding site, and a self-catalyzed hydrolysis ring as well as a W60d ring. Thrombin has Na ion-induced allosteric properties, and has two conformations of Na ion binding and Na ion dissociation in blood. These two conformations are interconvertible, and part of the interconversion mechanism is that the effector or substrate binds to a specific site of th...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573G01N27/26G01N21/76G01N33/531
CPCG01N21/76G01N27/26G01N33/531G01N33/573G01N33/577G01N2333/974
Inventor 泮锋纲丁俊杰施启尧
Owner 江苏三联生物工程股份有限公司
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