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Anti-heart fatty acid binding protein monoclonal antibody resistant to fatty acid interference and application thereof

A monoclonal antibody, protein technology, applied in the direction of anti-animal/human immunoglobulin, antibody, immunoglobulin, etc., to achieve the effect of good specificity, high detection sensitivity, good temperature stability

Active Publication Date: 2018-01-12
NANJING VAZYME MEDICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when preparing monoclonal antibodies for diagnosis, the antigens used to immunize animals and screen antibodies are all recombinantly expressed H-FABP proteins, so the recognition site of the obtained monoclonal antibodies may be located in the fatty acid binding region

Method used

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  • Anti-heart fatty acid binding protein monoclonal antibody resistant to fatty acid interference and application thereof
  • Anti-heart fatty acid binding protein monoclonal antibody resistant to fatty acid interference and application thereof
  • Anti-heart fatty acid binding protein monoclonal antibody resistant to fatty acid interference and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Expression purification and purity identification of embodiment 1 recombinant H-FABP protein

[0049] The prokaryotic expression plasmid containing the H-FABP gene sequence was transformed into Escherichia coli BL21(DE3), and cultured on LB plates. Pick a single colony from the plate and inoculate it into LB medium. After expanding the culture, when the OD600 value of the bacterial solution reaches 0.6-0.8, add IPTG with a final concentration of 1 mM to induce expression. After 4 hours, the bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation after the bacteria were disrupted by ultrasonication. The supernatant was purified by His-taq column and molecular sieve chromatography column, and the samples were collected and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein purity.

[0050] After electrophoresis, a target protein band with a molecular weight of about 15 kDa was observed on the ...

Embodiment 2

[0051] Example 2 Animal immunity and serum titer detection

[0052] The purified recombinant H-FABP protein with a concentration of 1mg / ml was emulsified with the same amount of Freund's complete adjuvant and Freund's incomplete adjuvant, and the emulsification method used the double syringe push method. 6-8 week specific pathogen-free grade female BALB / c mice were injected with immune antigen H-FABP emulsified in Freund's complete adjuvant to each mouse from the hock joint with 40 μg of antigen, 2 days later, with Freund's incomplete adjuvant The emulsified immune antigen H-FABP was injected into each mouse with 40 μg antigen again from the hock joint. Eight days later, blood was collected from the tail vein, centrifuged to obtain the supernatant, and the serum titer was detected by indirect ELISA with the pre-immune serum as a negative control. After the second immunization, the serum titer was as high as 2.0 or more after a million-fold dilution (Table 1). Select the serum...

Embodiment 3

[0055] Example 3 Cell Fusion, Positive Hybridoma Screening and Subcloning

[0056] 1. Preparation and screening of high-titer hybridoma cell lines

[0057] Sp2 / 0 myeloma cells in good growth state were selected and mixed with lymphocytes of immunized mice at a ratio of 1:2-1:3, and PEG1500 preheated at 37°C was used as a fusion agent. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum. After 5-7 days, observe the fusion effect and change the medium, and take the cell culture medium 3 days after the medium change. Use the serum of the patient within 6-12 hours of clinically confirmed myocardial infarction to coat the ELISA plate. The serum contains a high concentration of natural H-FABP protein. Use the indirect ELISA method to screen positive clones, and select the one with a higher ratio of positive value to cell number. The cells were subcloned multiple times, and finally a hybridoma cell library capable of stably secreting monoclonal antibodies ag...

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Abstract

The invention discloses a fatty acid interference-resistant anti-human heart-type fatty acid binding protein monoclonal antibody and application thereof. A hybridoma cell strain FP1 capable of stably secreting the fatty acid interference-resistant anti-human heart-type fatty acid binding protein monoclonal antibody is collected with the collection number of CCTCC NO: C2016199. Through measurement, the monoclonal antibody has the titer of 1: 10<6> and the affinity of 1*10<-13>M and binding of fatty acid to H-FABP protein does not affect the affinity. An H-FABP diagnostic kit prepared on the basis of the monoclonal antibody has very high sensitivity of 0.2 ng / ml, and can resist interference of fatty acids in blood to a detection result. After adoption of the H-FABP monoclonal antibody, a high-specificity and high-accuracy detection kit for early diagnosis of acute myocardial infarction can be developed, and a key reference is provided for early diagnosis of myocardial injury.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a monoclonal antibody against human heart fatty acid binding protein with strong anti-interference ability and application thereof. Background technique [0002] Acute coronary syndrome is a group of clinical syndromes based on the pathological basis of coronary atherosclerotic plaque rupture or invasion, followed by complete or incomplete occlusive thrombosis, including unstable angina and acute myocardial infarction. AMI). AMI is the main type of cardiovascular disease. In the past ten years, the incidence of AMI in my country has been on the rise and is close to the international average level. The onset of AMI is sudden, and the mortality rate in the acute phase is about 30%. At present, the best way to treat AMI in the world is early and rapid diagnosis and acute coronary intervention. [0003] The observation of electrocardiogram is a common method for dia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577A61K39/395A61P9/10C12R1/91
CPCA61K39/00C07K16/18C07K2317/92G01N33/577G01N33/6893G01N2333/47G01N2800/324
Inventor 曹林唐波牛英波徐晓昱杨晨辰
Owner NANJING VAZYME MEDICAL TECH CO LTD
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