Chemiluminescence immunity analysis detecting myocardium calcium protein T hypersensitization method for acridine ester and alkaline phosphatase

A chemiluminescence immunoassay and cardiac troponin technology, applied in the field of clinical immunoassay, can solve the problems of slow elimination rate and long duration.

Inactive Publication Date: 2008-07-23
天津天美生物技术有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

In addition, the elimination rate of cTnT in blood and lymph fluid is slow, and the

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Acridine ester DMAE-NHS [2',6'-dimethyl-4(N-succinimide-oxycarbonyl)phenyl-10-methyl-acridine-9-carboxylate-methoxysulfuric acid Salt] labeled monoclonal antibody:

[0019] 1. Preparation of two groups of monoclonal antibodies against cTnT were synthesized by the patentee. The specific method is detailed in the patent "Method for Detecting Cardiac Troponin T by Chemiluminescent Immunoassay, Example 1" (application number: 200610130403.3).

[0020] 2. Labeling method: take monoclonal antibody (2) 150μl×1.5g / L, and dialyze against carbonate buffer (pH 10.1, 0.1mol / L) overnight (4°C). Dissolve the acridinium ester DMAE-NHS in anhydrous dimethylformamide and make a 6.0mmol / L solution, take 8.0μl of this solution and add it to the dialyzed monoclonal antibody solution, stir and react at 4°C overnight, and add 5g / L solution the next day. 100 μl of L-lysine solution was continued for 1 hour to terminate the labeling reaction. The reaction solution was dialyzed against pH 6....

Embodiment 2

[0022] Method for detection of cTnT by chemiluminescence with acridinium ester bis-mab sandwich:

[0023] 1. Prepare the complex of monoclonal antibody (1) and biotin according to the above documents 1-6, and bind streptavidin to the Nunc plate. Because these methods have become conventional methods, they will not be repeated here.

[0024]2. Prepare the complex of monoclonal antibody (2) and acridinium ester DMAE-NHS according to Example 1.

[0025] 3. Detection of cTnT by sandwich chemiluminescence method of acridinium ester double monoclonal antibody: the complex of the serum sample to be tested (or cTnT standard 0.01-5.00ng / ml), monoclonal antibody (1) and biotin (1:2000) Add 50 μl of the complex (1:1500) of monoclonal antibody (2) and acridinium ester DMAE-NHS into the wells of the Nanc plate coated with streptavidin, incubate at 37°C for 60 minutes, and wash with PBST solution (note ) was washed 5 times, and the luminescent starter was added 0.15mol / LNaOH, 0.1%H 2 o ...

Embodiment 3

[0028] Alkaline phosphatase competitive CLIA method for detecting cTnT: The competitive CLIA method for detecting cTnT only needs one monoclonal antibody. Prepare cTnT-streptavidin complexes and alkaline phosphatase-biotin complexes according to the above-mentioned documents 1-6, and immobilize the capuzumab (1) on the solid phase carrier. These conventional methods are not repeated here. Add 50 μl each of the sample (or standard 0.01-8.00ng / ml), cTnT-streptavidin complex (0.6ng / ml), alkaline phosphatase-biotin complex (1:1500) Mix well and incubate at 37°C for 60 minutes. After the reaction, wash 5 times with PBST solution (the formulation is the same as in Example 2), and add the substrate CSPD of alkaline phosphatase and a total of 150 μl of enhancer after washing. React at room temperature for 30 minutes in the dark. The chemiluminescence detector of Beijing Hamamatsu Photonics Co., Ltd. was used to detect the wavelength of 470nm, and the cTnT content of the sample was ...

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Abstract

The invention relates to a chemical illumination immunity analysis method for checking human cTnT, which uses acridiniumester and/or alkaline phosphatase as label. The immunity reaction uses two-site immunoassay and/or competition law. The immunity reaction can use streptavidin-biotin two-site immunoassay to improve sensitivity. The invention uses NaOH and H2O2 as acridiniumester illumination initiating agent, uses 1, 2-dioxo cyclohexane derivative (adamantine derivative) as the illumination substrate of alkaline phosphatase, and uses fluorescein derivative and surface activator as illumination renforcing agent. The solid carrier is orifice plate, macromolecule polymer tube (ball) and ferriferrous oxide magnetic particles or the like.

Description

technical field [0001] The invention belongs to the field of clinical immune analysis, and establishes a method for detecting myocardial troponin T by chemiluminescent immunoassay using acridinium ester and alkaline phosphatase as markers, so as to diagnose acute myocardial infarction early, sensitively and specifically. In addition, the invention can also be used in laboratories for multiple researches on physiology, pathology, pharmacology, molecular biology and the like. technical background [0002] Cardiovascular disease is a common and frequently-occurring disease, and it is the primary disease that threatens human health. Acute myocardial infarction (AMI) is a common cardiovascular disease. Correct diagnosis and timely treatment are of great significance to save the dying myocardium, reduce the mortality rate in the acute phase, and improve the prognosis. In the diagnosis of AMI, the accuracy of commonly used electrocardiogram diagnosis is only 24-60% (Fesmire FM.et...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
Inventor 苏殿杰魏桂梅韩苏
Owner 天津天美生物技术有限公司
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