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ENO2 monoclonal antibody 1C5 as well as preparation method and application thereof

A monoclonal antibody, variable region technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of difficult recovery, unknown antibody sequence, poor cell state, etc., and achieve easy quality control. , good application value, widely used effect

Active Publication Date: 2022-05-13
SHAANXI MYBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the current detection of NSE, traditional monoclonal antibody-producing hybridoma cells are not easy to preserve, and the cell state will deteriorate if the time is too long, and it may even be difficult to recover, and the amount of antibody produced will decrease, and the sequence of the antibody is unknown, so it is impossible to change the antibody. Deep research

Method used

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  • ENO2 monoclonal antibody 1C5 as well as preparation method and application thereof
  • ENO2 monoclonal antibody 1C5 as well as preparation method and application thereof
  • ENO2 monoclonal antibody 1C5 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Preparation of ENO2 protein

[0080] Step 1. Construction of ENO2 prokaryotic expression vector

[0081]1. Check the human ENO2 gene sequence (SEQ ID NO.19) from the GenBank sequence database, the sequence number is NM_001975.3, synthesize the gene sequence between NdeI and NotI of the pET-32a vector, and connect the target gene ENO2 at the same time between EcoRI and NotI of the pGEX 4t-1 vector;

[0082] 2. Design the primers and amplify the target band by PCR.

[0083] Upstream primer ENO2(4t-1)-EcoRI-F (SEQ ID NO.20):

[0084] ATCTGGTTCCGCGTGGATCCCCGGAATTCatgtccatagagaagatctgggc;

[0085] Downstream primer ENO2(4t-1)-NotI-R (SEQ ID NO.21):

[0086] CAGTCAGTCACGATGCGGCCGCTCGAGtcacagcacactgggattacgga.

[0087] PCR amplification system (50μl): template 50ng, ENO2(4t-1)-EcoRI-F (10μM) 1μl, ENO2(4t-1)-EcoRI-R (10μM) 1μl, FastPfu DNA Polymerase (2.5units) 1μl, 5×FastPfu buffer 10μl, 2.5mM dNTP 4μl and the rest Nuclease-free Water;

[0088] PCR amplificati...

Embodiment 2

[0099] The acquisition of embodiment 2 hybridoma cells

[0100] Step 1. Immunization of mice with ENO2 antigen

[0101] 1. Mix 40 μg of purified ENO2-his antigen with complete Flureund's adjuvant 1:1;

[0102] 2. Take 5 female Balb / c mice aged 6-8 weeks, and inject 100 μL (40 μg antigen) of the mixed antigen into the left calf muscle; perform the second immunization three weeks later, 40 μg antigen and incomplete Fluorine The adjuvant was mixed 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the muscle of the right hind calf;

[0103] Step 2. ELISA detection of antibody production in mice

[0104] Three weeks after the second immunization, the mouse tail blood was collected, the serum was collected by centrifugation, and the antibody production of the mice was detected by ELISA. The specific steps are as follows:

[0105] ①Coat the ELISA plate with purified ENO2-GST protein, 100ng / well, overnight at 4°C, coating solution: 25mL carbonate buffer, pH 9.6...

Embodiment 3

[0127] Example 3 Preparation of Monoclonal Antibody from Ascites and Purification of Monoclonal Antibody

[0128] Step 1. Preparation of monoclonal antibody from ascites

[0129] 1. Inject 300 μl of ascites adjuvant into the peritoneal cavity of 12-week-old Balb / c mice.

[0130] 2. Two weeks later, the hybridoma cells were cultured to the best cell activity state, and the cell number was adjusted to about 1×10 6 Each / 100ul, inoculate 100ul hybridoma cells into the peritoneal cavity of mice injected with ascites adjuvant.

[0131] 3. Collect ascites after 7-10 days.

[0132] Step 2. Monoclonal Antibody Purification

[0133] 1. The ascites was diluted with PBS pH7.4, centrifuged to obtain the supernatant, and purified by protein G affinity chromatography.

[0134] 2. Balance: Equilibrate the purification column with 0.4M PB buffer (pH 7.0);

[0135] 3. Column loading: Pass the diluted ascites supernatant slowly through the column to ensure that the antibody is better bound ...

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Abstract

The invention provides an ENO2 monoclonal antibody 1C5 as well as a preparation method and application thereof, and relates to the technical field of monoclonal antibodies. The invention discloses a complementary determining region sequence of variable regions of a heavy chain and a light chain of the monoclonal antibody 1C5, the variable region sequence can be constructed on an antibody expression vector based on the sequence, and a recombinant monoclonal antibody is obtained by transfecting cell expression. The monoclonal antibody 1C5 vector obtained by the invention is easy to store, and the quality control in the antibody production process is easy; the monoclonal antibody 1C5 disclosed by the invention can recognize the ENO2 protein, has biological activity, can be used for ENO2 antigen detection and other scientific researches, and has very good application value and very important scientific research guiding significance.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibodies, and in particular relates to an ENO2 monoclonal antibody 1C5 and its preparation method and application. Background technique [0002] Enolase is a key enzyme in the process of glycolysis in organisms. In vertebrates, there are three isozymes of enolase: α, β and γ. α-enolase (ENO1), also known as non-neuralenolase (NNE), exists in many tissues; β-enolase (ENO3), also known as muscle-specific enolase (muscle-specific enolase, MSE ), skeletal muscle enolase (skeletalmuscleenolase), almost only found in muscle tissue; γ-enolase (ENO2) is also known as neuronal enolase (neuraleno-lase), neuron-specific enolase NSE), mainly found in neurons and neuroendocrine tissues. The active forms of Enolase are all dimers, that is, composed of two subunits. There are currently known combinations of five forms: αα, ββ, γγ, αβ and αγ. [0003] Enolase 2 (ENO2), also known as NSE, mainly exists in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N15/85C12N5/10C12P21/08G01N33/577G01N33/573
CPCC07K16/40C12N15/85G01N33/577G01N33/573C07K2317/56C07K2317/565C07K2317/567G01N2333/988G01N2800/2871G01N2800/52
Inventor 闫亚平郝文斌张亚剑穆瑜王璐刘龙月李科
Owner SHAANXI MYBIOTECH CO LTD
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