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Reagent for detecting acute myocardial infarction by immunological method and test strip

A technology for acute myocardial infarction and test strips, applied in biochemical equipment and methods, biological testing, anti-animal/human immunoglobulin, etc., can solve problems such as the use of other indicators, long detection time, and complicated operations, and achieve The effect of improving sensitivity and specificity and improving diagnostic accuracy

Active Publication Date: 2010-08-18
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above studies suggest that H-FABP has certain advantages as a biochemical marker for the early diagnosis of AMI, but how to use it in conjunction with other indicators has not been reported
[0008] In addition, in terms of AMI detection methods, the current commonly used methods in China are enzyme-linked immunoassay and chemiluminescence. This method requires specific instruments, high cost, complicated operation, and long detection time. It is not suitable for self-examination or initial detection of AMI patients. Rapid screening and diagnosis in primary hospitals and emergency rooms

Method used

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  • Reagent for detecting acute myocardial infarction by immunological method and test strip
  • Reagent for detecting acute myocardial infarction by immunological method and test strip
  • Reagent for detecting acute myocardial infarction by immunological method and test strip

Examples

Experimental program
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Effect test

Embodiment 1

[0060] The preparation of embodiment 1.H-FABP, cTnI recombinant antigen

[0061] Preparation of H-FABP recombinant antigen:

[0062] According to the human H-FABP DNA sequence published by GENBANK, two PCR primers containing restriction endonuclease sites at both ends were designed and synthesized. The sequence is

[0063] 5'-AGCTGGATCCATGGTGGACGCTTTCCTGGGC-3';

[0064] 5'-CTAGGAATTCTGCCTCTTTCTCATAAGTGCG-3'.

[0065] The cDNA sequence of H-FABP was amplified from the heart tissue by RT-PCR, inserted into the expression vector pET16b, and after the gene sequence was confirmed to be correct by sequencing, it was transferred into E.coli BL / 21 (LysE) strain, induced by IPTG, and expressed The H-FABP was separated and purified by Ni-Agarose affinity chromatography column to obtain H-FABP recombinant antigen. The specific operation was carried out according to the method taught in "Molecular Cloning" and the product manual.

[0066] Preparation of cTnI recombinant antigen:

[0...

Embodiment 2

[0071] The preparation of embodiment 2.H-FABP, cTnI monoclonal antibody

[0072] Immunize each BALB / c mouse with intraperitoneal injection of 100 μg of the suspension of the above-mentioned purified recombinant protein + Freund’s complete adjuvant for the first time, and then inject 100 μg of the suspension of purified recombinant protein + Freund’s incomplete adjuvant every 1 month 1 time, a total of 3 times, and 3 days before the spleen was killed for cell fusion, the antigen was directly injected into the tail vein to boost the immunization once.

[0073] Production and screening of hybridoma cells The fusion of SP2 / 0 tumor cells and splenocytes of immune mice and the cloning of hybridoma cells were carried out according to the routine methods of our laboratory. Coat the ELISA plate with the above-mentioned purified recombinant protein antigen, use the indirect ELISA method to detect and screen the positive wells of the hybridoma cell culture supernatant, clone and culture ...

Embodiment 3

[0085] Example 3. Preparation of colloidal gold-labeled antibody solution

[0086] Prepare H-FABP, cTnI-specific antibody-labeled colloidal gold gold-labeled probe solution and gold-labeled antibody pad:

[0087] Colloidal gold is prepared by citric acid reduction method: 0.01% HAuCl4 (Shanghai Trial Brand purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.) aqueous solution is boiled with a mass percentage concentration, and 2 mL of trisodium citrate with a mass percentage concentration of 1% is added under stirring Aqueous solution, continue to boil until the liquid is dark red to obtain a colloidal gold solution.

[0088] Determine the saturation of the colloidal gold-coupled antibody: adjust the pH value to 8.5 with 0.1M K2CO3 solution, prepare a 96-well microtiter plate, and dilute the specific antibody against H-FABP and cTnI with 0.05M boric acid buffer in the well , make a dilution gradient, add the same volume of colloidal gold and mix well, then add the sam...

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Abstract

The invention relates to a medical diagnostic reagent, in particular to a quick detection reagent for the early diagnosis of acute myocardial infarction and a test strip. The invention provides a double-index united detection reagent containing a specific antibody resisting a human heart-type fatty acid binding protein H-FABP and a human cardiac troponin cTnI. The invention also provides a colloidal gold labeling immunological chromatographic test strip containing the specific antibody resisting the H-FABP and the cTnI, which is used for quickly detecting the acute myocardial infarction. The double-index united detection reagent can carry out early diagnosis on a patient having the acute myocardial infarction, can also prevent the missed diagnosis of a patient having long-time chronic myocardial infarction with mild symptoms and better solves the influence caused by a difference existing in the detection time. The colloidal gold labeling immunological chromatographic test strip provides a quick, convenient, cheap and practical detection tool for the early diagnosis of the acute myocardial infarction, is hopefully used for hospitals at all levels and can also be used for the self monitoring of the patient.

Description

technical field [0001] The invention relates to a medical diagnostic reagent, in particular to a diagnostic reagent and a test strip for rapidly diagnosing acute myocardial infarction. Background technique [0002] Acute myocardial infarction (AMI) is a common emergency and critical disease in internal medicine. It has become a disease that seriously affects human health. The golden time for thrombolytic therapy and interventional surgery is 1 to 6 hours after the onset of the disease. Rapid diagnosis of early AMI within 6 hours of the onset It is a key link in deciding on treatment. According to the recommendation of the World Health Organization (WHO): AMI can be diagnosed if two of the three indicators of typical chest pain, electrocardiogram changes and abnormal myocardial enzymes meet. However, in my country, about 25% of AMI patients are asymptomatic, and about 30% of cases have no typical angina pectoris; the average accuracy of using electrocardiogram to diagnose AM...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/551C07K16/18C12N5/20
Inventor 张云涛席仲兴杨福军彭林峰张正雷张甲菊
Owner LANZHOU INST OF BIOLOGICAL PROD
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