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Method for chemiluminescence immunity analysis and detection myocardium calcium protein T

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

Inactive Publication Date: 2008-06-25
天津天美生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the elimination rate of cTnT in blood and lymph fluid is slow, and the increase of cTnT can last for 2-3 weeks during AMI, which is longer than that of cTnI

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Extraction and purification of cTnT and preparation of its monoclonal antibody. References [Jin J-P, J.Biol.Chem.1988, 263(15): 7309; Fu Chaoping, Journal of Cellular and Molecular Immunology, 1996, 12(1): 39; Zhou Guohua, etc., Chinese Journal of Physiology, 2004, 6 (2): 198; Zhang Haizhu et al., Journal of Zhengzhou University (Medical Edition), 2003, Issue 03; Li Zhiliang, Fu Chaoping, Progress in Biochemistry and Biophysics, 1996, Issue 5〕, fresh myocardium without heart disease The tissue was homogenized without salt, extracted with high salt at 70°C, salted out with ammonium sulfate, and subjected to DEAE cellulose column chromatography to obtain pure cTnT. 100g myocardial tissue obtained 4.5mg cTnT. The cTnT was a band by gel electrophoresis, and the second peak of DEAE fiber column chromatography was identified as the activity peak of cTnT by immunoblotting.

[0018] BALB / c mice were immunized with cTnT. For the first time, 35 μg of cTnT was injected intraper...

Embodiment 2

[0021] The preparation method of the horseradish peroxidase luminescent system: the concentration of the luminescent substrate diacylhydrazine derivative (preferably luminol) is 1.0mMol / L. Compound oxidant: sodium perborate-H 2 o 2 Each is 1.0mMol / L. Composite enhancer: 0.05mMol / L for each of p-hydroxycitric acid and p-iodophenol. Adjust the pH with 0.1Mol / L Tris-HCl buffer at pH 8.60.

Embodiment 3

[0023] Establishment of a one-step chemiluminescent immunoassay method for the detection of cTnT by avidin-biotin double monoclonal antibody sandwich:

[0024] 1. Prepare the complex of monoclonal antibody (1) and biotin and the complex of monoclonal antibody (2) and horseradish peroxidase according to the aforementioned literature, and bind streptavidin to the Nunc plate.

[0025] 2. Establish a one-step CLIA method for cTnT detection with avidin-biotin double monoclonal antibody sandwich. Add 50 μl each of the serum to be tested (or cTnT standard), the complex of monoclonal antibody (1) and biotin, and the complex of monoclonal antibody (2) and horseradish peroxidase into the streptavidin-coated In the wells of the Nanc plate, incubate at 37°C for 60 minutes, wash 3 times with PBST, add luminol substrate solution (see Example 2 for specific preparation), detect the luminescence value at 425nm with a chemiluminescence immunoassay analyzer, and calculate it by computer softwar...

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PUM

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Abstract

The invention relates to an analysis method for using the means of chemiluminescence immunoassay to detect cardiac troponins T(cTnT).

Description

technical field [0001] The invention belongs to the field of clinical blood immune analysis, and establishes a chemiluminescence immunoassay method for detecting cardiac troponin T to diagnose acute myocardial infarction early, sensitively and specifically. In addition, the present invention can also be used in laboratories for multiple studies on physiology, pathology, and pharmacology. 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 dying myocardium, improve prognosis and reduce acute mortality. In the biochemical diagnosis of AMI, creatine kinase (CK) and its isoenzymes (CK-MB), lactate dehydrogenase (LDH), etc. are currently commonly used myocardial enzymes. However, myocardial enzymes are not unique to the heart, and th...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/53G01N33/577G01N21/76
Inventor 苏殿杰王仁芝魏桂梅韩苏
Owner 天津天美生物技术有限公司
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