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Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof

A cardiac troponin, monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, microorganism-based methods, etc.

Inactive Publication Date: 2011-01-12
INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the research work is still in the laboratory stage, so far no commercialized recombinant human cardiac troponin I has come out

Method used

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  • Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof
  • Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof
  • Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] step:

[0029] 1. Design of PCR primers:

[0030] Primers were designed according to the human cardiac troponin I gene sequence provided by the Gene Bank database on the NCBI website. NdeI and BamHI endonuclease sites were inserted at the 5' ends of the upstream and downstream primers, respectively, and the 2nd and 4th positions of the upstream primers were Site-directed mutagenesis of the third degenerate base of the first codon, using Primer Premier5.0 and DNAMAN software to analyze the primers.

[0031] 2. RT-PCR amplification of the target gene:

[0032] The annealing temperature was set at 56.0°C, the amplified product was detected by 12g / L agarose gel electrophoresis, and the target gene fragment was recovered by cutting the gel.

[0033] 3. Construction and identification of pMD19-T / rhcTnI cloning plasmid:

[0034] Inoculate the target gene fragment recovered from the gel cutting with the pMD19-T vector (product of Dalian TAKARA Company) and transform it into ...

Embodiment 2

[0091] Preliminary Clinical Application Effect Experiment of Cardiac Troponin I

[0092] Principle: The double-antibody sandwich method is used to measure the level of human cardiac troponin I in the specimen, and the purified human cardiac troponin I antibody is used to coat the microwell strip to make a solid-phase antibody, which is sequentially poured into the microwells coated with monoclonal antibody. Add standard or sample, then combine with HRP-cTnI antibody to form antibody-antigen-enzyme-labeled antibody complex, add TMB to develop color, it is blue, and finally yellow under the action of acid, the color depth is related to the cTnI content in the sample If there is a positive correlation, use a microplate reader to measure the absorbance at a wavelength of 450nm to determine its content.

[0093] The microwell plate strip (coating concentration 5ug / ml) coated with the monoclonal antibody of cTnI obtained by the present invention replaces the microwell plate strip of...

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Abstract

The invention relates to an anti-human cardiac troponin I hybridoma cell line, with the preservation number of CGMCCNO. 3951, and a monoclonal antibody secreted from the anti-human cardiac troponin I hybridoma cell line CGMCC3951. The class and subclass of immunoglobulin of the monoclonal antibody are respectively IgG and IgG3 and the monoclonal body is specifically combined with human cardiac troponin I; the potency is 1:16000 and the affinity constant is 1.08x10 to 9mol / L. By applying the monoclonal antibody of the invention to clinical diagnosis, the study on complete localization of diagnosis kit for cardiac troponin I is tremendously promoted, correctness of clinical diagnosis and prognosis for cardiovascular diseases is enhanced while suffering of patients and death rate are both reduced, thus prominent economical and social benefits are obtained.

Description

technical field [0001] The invention relates to a biological product for in vitro experiments characterized by antibodies, more specifically, a monoclonal antibody with high specificity against human cardiac troponin I (cTnI) and its preparation method and application. Background technique: [0002] Early diagnosis and timely treatment of acute myocardial infarction (AMI) are the key to reducing mortality. The determination of myocardial tissue-specific markers is the main index for AMI diagnosis, monitoring of disease course and evaluation of prognosis. Traditionally used early markers are myoglobin (Mb), lactate dehydrogenase (LDH) isoenzyme, creatine phosphokinase isoenzyme (CK-MB), etc. Clinical practice has proved that most of these myocardial injury markers have specificity. However, there are defects such as weak sex, late rising time and relatively short duration. In recent years, cardiac troponin I (cTnI) has shown its special superiority in the diagnosis of AMI a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 王德芝张春明杨磊郝建秀李玉彬宋娜玲贺欣刘鉴峰佘义王沂冯丽娜
Owner INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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