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A pair of monoclonal antibodies binding to human myoglobin with high specificity and high affinity and application thereof

A technology of monoclonal antibody and human myoglobin, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, material inspection products, etc., to achieve good detection specificity and sensitivity, and large linear range

Active Publication Date: 2018-05-01
广州普锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, foreign countries have successfully developed a double-antibody sandwich Elisa kit that can be used to detect human myoglobin, but it is still the development of domestic kits that can compete with imported kits and break the monopoly of foreign products. Striving Direction

Method used

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  • A pair of monoclonal antibodies binding to human myoglobin with high specificity and high affinity and application thereof
  • A pair of monoclonal antibodies binding to human myoglobin with high specificity and high affinity and application thereof
  • A pair of monoclonal antibodies binding to human myoglobin with high specificity and high affinity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of Antigen

[0048] Human Myoglobin (Human Myoglobin), purchased from ABCam Company, product number ab77876, its concentration is 2.4 mg / mL, is a natural human myoglobin molecule with a molecular weight of 16.7 kDa and consists of 154 amino acid residues. The main function of human myoglobin is to transport and store oxygen in muscle cells. When myocardial infarction occurs, it is one of the earliest markers that appear in the blood.

Embodiment 2

[0049] The preparation of embodiment 2 monoclonal antibody

[0050] 1. Materials used in this embodiment

[0051] (1) Reagents and animals: human myoglobin standard antigen, anti-human myoglobin standard antibody, and human myoglobin Elisa detection kit were purchased from ABCam Company; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company of the United States, PRMI1640 medium, fetal bovine serum, HAT, HT, polyethylene glycol (PEG, Mw4000) are all products of Gibco Company of the United States; low cross-reaction diluent and enzyme-labeled antibody protection solution are purchased from Candor Bioscience Company of Germany ; Mouse myeloma cells (SP2 / 0) were passaged and preserved in our laboratory; SPF-grade BALB / c pure-line female mice, aged 6-8 weeks, were purchased from the Animal Center of Southern Medical University; horseradish peroxidase Labeling kits (type A, type B) were purchased from Taitianhe Biotechnology Co., Ltd.; normal...

Embodiment 3

[0099] Characteristic identification of embodiment 3 monoclonal antibody

[0100] 1. Determination of Ig class and subclass

[0101] The commercially available mouse mAb typing kit was used to identify the Ig class and subclass of 10 strains of anti-human myoglobin monoclonal antibodies after affinity purification, and all operations were performed strictly according to the instructions of the kit.

[0102] The results are shown in Table 1, wherein 2M1, 5M7, 10M4, 2M5 and 5M11 are all IgG1, 3M4 and 2M11 are IgG2b, and 10M1 and 3M7 are IgG3.

[0103] 2. Monoclonal antibody titer determination

[0104] The titer of the nine affinity purified antibodies was determined by indirect Elisa.

[0105] Dilute human myoglobin standard antigen to 300ng / ml with coating solution, add 100μl to each well, and incubate overnight at 4°C. Wash with PBST (0.05% Tween content) three times for 3 minutes each time, then block with 5% skimmed milk powder at 37°C for 1 hour, wash with PBST three ti...

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Abstract

The invention discloses a pair of hybridoma cell lines 3M4 and 5M7 secreting monoclonal antibodies with high specificity and high affinity binding to human myoglobin (both were preserved in China Center for Type Culture Collection on March 25, 2015, and the preservation numbers are respectively CCTCC C201529 and CCTCC C201530), and their secreted monoclonal antibodies 3M4 and 5M7. The pair of antibodies has high specificity and high affinity, can specifically bind to different epitopes of human myoglobin, forms a double-antibody sandwich mode, and realizes the quantitative detection of myoglobin in human serum, with good detection specificity and sensitivity . The detection sensitivity is similar to that of imported kits, but the double-antibody sandwich Elisa method established by the present invention has a larger linear range, which can be used clinically for the detection of myoglobin in human serum, and has important guidance for the early diagnosis of myocardial infarction Significant, with commercial application value.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a pair of monoclonal antibodies binding to human myoglobin with high specificity and high affinity and applications thereof. Background technique [0002] For a long time, cardiovascular diseases, especially acute myocardial infarction (AMI) have been the culprit that seriously affects human health. Once myocardial infarction occurs, if a clear diagnosis and effective treatment are not obtained in a short period of time, the human heart muscle will be damaged due to Irreversible necrosis due to ischemia and hypoxia. Therefore, early and timely diagnosis of acute myocardial infarction is particularly important to reduce mortality. The discovery of serum myocardial injury markers provides an important basis for the diagnosis of acute myocardial infarction. [0003] The diagnosis of acute myocardial infarction using myocardial injury markers has a history of more than...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 向军俭杨全利黄建芳
Owner 广州普锐生物科技有限公司