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Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit

A monoclonal antibody and detection kit technology, applied in the field of in vitro diagnostic medical testing, can solve the problems of low sensitivity, difficulty in controlling batch-to-batch differences, unfavorable promotion of myoglobin detection, etc., achieve high detection sensitivity, increase reaction signal, and result good stability effect

Inactive Publication Date: 2017-02-22
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although there are registered and approved myoglobin chemiluminescence immunoassay assay kits in the Chinese market, most of them are imported reagents with high cost, and the preparation methods and related materials of the kits are kept as confidential information and have not been made public. It is not conducive to the promotion of myoglobin detection in community primary hospitals
Domestic myoglobin kits have poor reproducibility, difficult to control batch-to-batch differences, low sensitivity, and poor clinical application prospects

Method used

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  • Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit
  • Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit
  • Myoglobin monoclonal abzyme marking compound and preparation method thereof and detection test kit

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preparation example Construction

[0065] Correspondingly, the present invention also provides a method for preparing the myoglobin monoclonal antibody enzyme-labeled complex, the method comprising the following steps:

[0066] 1) cross-linking myoglobin monoclonal antibody with enzyme to prepare antibody enzyme marker;

[0067] 2) adding the abzyme-labeled substance obtained in step 1) into the colloidal gold solution, thereby obtaining the myoglobin monoclonal abzyme-labeled complex.

[0068] In a specific embodiment, the method includes:

[0069] a) the myoglobin monoclonal antibody and the enzyme are cross-linked by the glutaraldehyde method to prepare antibody enzyme markers;

[0070] b) adding trisodium citrate in gold chloride solution to prepare colloidal gold solution;

[0071] c) adding the abzyme marker reacted in step a) to the colloidal gold solution, mixing evenly, and adding the blocking solution;

[0072] d) Preserving the antibody marker treated in step c) in the magnetic particle preservati...

Embodiment 1

[0143] Example 1. Preparation of myoglobin monoclonal antibody enzyme-labeled complex

[0144] 1) Dissolving alkaline phosphatase in a final concentration of 5% glutaraldehyde solution, so that the concentration of alkaline phosphatase is 10 mg / mL;

[0145] 2) Stand overnight at room temperature;

[0146] 3) Dialysis with normal saline (volume ratio greater than 1:200) for at least 4 hours;

[0147] 4) Take out the dialyzed alkaline phosphatase, add myoglobin monoclonal antibody according to the mass ratio of 1:1, and mix well;

[0148] 5) Dialyze in 50mM pH 9.6 sodium carbonate-sodium bicarbonate buffer overnight;

[0149] 6) Take out the dialysate, add 1M glycine solution according to the volume ratio of 1:1000, mix well, and stop the reaction for 2 hours;

[0150] 7) Add an equal volume of saturated ammonium sulfate and store at 2-8°C for 2 hours;

[0151] 8) Centrifuge at 4000rpm to remove the supernatant, and dissolve the precipitate in 50mM pH 7.40 phosphate buffer;

...

Embodiment 2

[0157] Example 2. Sensitivity detection of myoglobin monoclonal antibody enzyme-labeled complex

[0158] Using the myoglobin monoclonal antibody enzyme-labeled complex obtained in Example 1, the sensitivity detection was carried out as described in the materials and methods, and the results are shown in the following table:

[0159]

[0160] Note:

[0161] (1) Traditional myoglobin monoclonal antibody enzyme markers;

[0162] (2) Using colloidal gold-coated myoglobin monoclonal antibody enzyme-labeled complex.

[0163] As can be seen from the above table, using the colloidal gold-coated myoglobin monoclonal antibody enzyme-labeled complex of the present invention, its sensitivity (S / N value) has increased by 1.92 times than traditional myoglobin monoclonal antibody enzyme-labeled substances . In addition, the functional sensitivity of its detection is 0.5 ng / mL, which is significantly lower than that of traditional myoglobin monoclonal antibody enzyme markers. To sum up...

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Abstract

The invention discloses a myoglobin monoclonal abzyme marking compound. The myoglobin monoclonal abzyme marking compound is composed of a myoglobin monoclonal abzyme marker and colloidal gold, wherein the myoglobin monoclonal abzyme marker marks the colloidal gold. The invention further provides a preparation method of the myoglobin monoclonal abzyme marking compound and a myoglobin detection test kit containing the myoglobin monoclonal abzyme marking compound. The use of the myoglobin monoclonal abzyme marking compound can significantly improve the sensitivity of myoglobin detection and ensure the specificity of a reagent.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic medical testing. Specifically, the invention relates to a myoglobin monoclonal antibody enzyme-labeled complex, a preparation method thereof and a myoglobin detection kit comprising the same. Background technique [0002] Myoglobin (Myoglobin, MYO) is a binding protein composed of a peptide chain and a heme prosthetic group, a heme protein that exists in large amounts in striated muscle (skeletal muscle and cardiac muscle) cells. When cardiac or skeletal muscle is damaged, myoglobin is released into the vascular system due to rupture of the cell membrane and can be detected in the blood. [0003] The concentration of myoglobin in the blood of patients with acute myocardial infarction (AMI) increases rapidly 1-3 hours after onset, and the myoglobin concentration in almost all AMI patients increases at 12 hours. Therefore, no increase in myoglobin within 6 to 12 hours of the onset of chest pain ...

Claims

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Application Information

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IPC IPC(8): G01N33/72G01N33/577G01N33/535
CPCG01N33/72G01N33/535G01N33/577G01N2800/324
Inventor 杭红陈超朱宗泽乔元彪彭珊李基
Owner SHANGHAI KEHUA BIO ENG
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