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Troponin I resisting monoclonal antibody and application thereof

A monoclonal antibody, troponin technology, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of not being able to actively prompt myocardial infarction symptoms, difficult diagnosis, etc., and achieve high diagnosis. Consistency, good stability and high sensitivity

Active Publication Date: 2011-04-27
上海贝西生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(6) Surgical injury assessment: Postoperative myocardial infarction is a common cause of postoperative death, with a mortality rate as high as 36% to 70%. However, it is difficult to diagnose, because surgical patients are often under anesthesia and sedation, and cannot actively prompt myocardial infarction symptoms

Method used

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  • Troponin I resisting monoclonal antibody and application thereof
  • Troponin I resisting monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Preparation of anti-TnT I monoclonal antibody

[0017] 1. Animal immunity

[0018] Take female BALB / c mice aged 6 to 8 weeks, and inject pure troponin I with a concentration of 5 mg / ml for the first immunization, and inject 1 ml in total through the abdominal cavity and under the armpit of the limbs; do the same method every 2 weeks Booster immunization once, a total of 3 immunizations, on the 4th day after the last immunization, blood was collected from the eyeballs of the mice after three immunizations, the serum was separated by centrifugation, and the mice with high titer were selected by ELISA method to prepare for fusion.

[0019] 2. Preparation of hybridoma cell lines

[0020] The splenocytes from the mice that had completed the immunization process were prepared to be fused with mouse myeloma cell SP2 / 0, and the feeder cells were prepared the day before the fusion; the mouse splenocytes were aseptically taken during fusion, and the splenocytes were m...

Embodiment 2

[0026] Example 2: Specific identification of monoclonal antibodies

[0027] Materials: Troponin C, Troponin T, Myoglobin, Creatine Kinase (CK), Creatine Kinase Isoenzyme (CK-MB).

[0028] Methods: ELISA method was used for detection, and troponin I was used as positive control.

[0029] Results: Troponin C, troponin T, myoglobin, and creatine kinase MB (CKMB) were all negative.

[0030] Explanation: The anti-troponin I monoclonal antibody of the present invention is highly specific to troponin I.

Embodiment 3

[0031] Example 3: Cloning and sequencing of variable region sequences of monoclonal antibodies

[0032]Using 5'RACE (Rapid Amplification of cDNA Ends, rapid amplification of cDNA ends) technology, the variable region sequence of functional antibody was cloned from the hybridoma cell line 5G9A10E3 secreting anti-troponin I monoclonal antibody. The steps can be briefly described as follows: an antisense gene-specific primer (GSP1) is used to synthesize the first strand of cDNA; A synthetic homopolynucleotide anchor sequence is added to the 3' end. The cDNA is amplified using a second nested gene-specific primer (GSP2) and an anchor primer that anneals to the homopolynucleotide tail. The specific experimental process is as follows:

[0033] Material:

[0034] -Anti-Troponin I monoclonal antibody secreted by the hybridoma cell line 5G9A10E3

[0035] - E. coli Top10 strains

[0036] -pGEM-T Easy Cloning Vector

[0037] Primer design:

[0038] According to the characteristics...

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Abstract

The invention discloses a troponin I resisting monoclonal antibody. An amino acid residue sequence of a light chain of the antibody is shown in SEQ ID No.1 and the amino acid residue sequence of a heavy chain is shown in SEQ ID No.2. The antibody is generated by a hybridoma cell line 5G9A10E3 with a preservation number of CCTCC NO.C201059 through secretion. The troponin I resisting monoclonal antibody can be applied to preparing various detection kits of the troponin I. A detection kit prepared by using the monoclonal antibody and a colloid gold immunochromatographic assay can ensure that the detection sensitivity reaches 97.74 percent, the detection specificity reaches 94.74 percent and the detection accuracy reaches 96.72 percent, and ensure that the Kappa detection value reaches 0.927 and far more than 0.75, has higher diagnosis consistency with the clinical diagnosis and can be used for auxiliary diagnosis for the acute myocardial imfarction disease.

Description

technical field [0001] The invention relates to an anti-troponin I monoclonal antibody and its application, belonging to the field of biotechnology. Background technique [0002] Cardiac troponin I (cTnI) is a unique protein marker in the myocardium, with a molecular weight of 22.5kD. In the heart, TnI, together with TnC and TnT, forms the troponin complex, which plays a pivotal role in the propagation of intercellular calcium signals when the myocardium contracts and relaxes. Biochemical indicators of early myocardial injury are mainly limited to myoglobin and CK-MB. However, not only does CK-MB exist in blood for a short period of time (1 to 4 days), but CK-MB also exists in skeletal muscle and brain. When skeletal muscle and brain are injured, CK-MB will increase significantly. Since the 1990s, a large number of clinical studies on troponin have shown that troponin has a high degree of specificity and sensitivity to myocardial injury. The National Association of Clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/577C12R1/91
Inventor 黄桂民周季余黄波
Owner 上海贝西生物科技有限公司
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