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Human agrin antigen, human agrin antibody detection kit, preparation method and application thereof

A technology of antigen and carrier, applied in the field of biopharmaceuticals, can solve the problem of not being detected, and achieve the effect of strong specificity, good stability and high sensitivity

Active Publication Date: 2022-05-24
WUHAN EASYDIAGNOSIS BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, AChR antibodies are undetectable in approximately 20% of MG patients
At present, there is no relevant report on the human Agrin antibody detection kit

Method used

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  • Human agrin antigen, human agrin antibody detection kit, preparation method and application thereof
  • Human agrin antigen, human agrin antibody detection kit, preparation method and application thereof
  • Human agrin antigen, human agrin antibody detection kit, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of recombinant expression plasmid and engineering bacteria

[0055] 1. Agrin is a heparan sulfate proteoglycan composed of multiple domains, including 9 protein kinase inhibitor domains, 4 epidermal growth factor-like domains and 1 adhesion molecule G homology domain. The mature Agrin protein is composed of 2038 amino acids. In this study, the amino acid sequence of Agrin protein was analyzed including hydrophilicity and surface accessibility, and combined with its spatial conformation and the modification characteristics of each domain, fragments of 7 regions of mature Agrin protein were selected to obtain them respectively. After a series of studies, the inventors obtained seven human Agrin antigens, Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864.

[0056] 2. PCR amplification of human Agrin antigen gene

[0057] 1. The DNA sequences of the 7 fragments were respectively subjected to gene synthesis, and the p...

Embodiment 2

[0066] Example 2 Expression and purification of antigen

[0067] 1. Use the constructed recombinant protein expression engineering bacteria to conduct the induction expression experiment. Inoculate 7 recombinant bacteria in 600ml of LB broth (components: 10g sodium chloride / liter, 10g peptone / liter and 5g yeast extract / liter), shake the bacteria at 37 degrees and 200RPM to OD600 to 0.6-0.8, press IPTG at a concentration of 24 mg / ml was added at 1:1000 for induction for 4 hours. Centrifuge, harvest bacteria, and prepare for purification.

[0068] 2. The selected filler for purification is GE's Ni Sepharose (Item No. 17-0729-10), and the following solutions are prepared according to its instructions:

[0069] Loading Buffer A: 0.5M NaCl + 20mM Na 2 HPO 3 +10 mM imidazole.

[0070] Binding buffer B: 0.5M NaCl + 20mM Na 2 HPO 3 +20mM imidazole

[0071] Elution buffer C: 0.5M NaCl, 20mM Na 2 HPO 3 , 500mM imidazole;

[0072] and the solution used to purify inclusion bodi...

Embodiment 3

[0078] Embodiment 3 Human Agrin antibody detection kit and method of use

[0079] 1. Coated ELISA plate

[0080] (1) Coating solution: NaCl 8.5g, Na 2 HPO 4 ·12H 2 O 30.8g, KH 2 PO 4 2.2g, add ddH 2 O to 1000ml, adjust pH to 7.4.

[0081] (2) Lotion for coating: NaCl 8.0g, KH 2 PO 4 0.24g, Na 2 HPO 4 ·12H 2 O 2.9g, KCl 0.2g, TWEEN200.5ml, add to ddH 2 O to 1000ml, adjust to PH7.4.

[0082] (3) Coating method: 7 Agrin antigen fragments were respectively coated in the microplate wells with 0.1M PBS (PH7.4) coating buffer, overnight at 4 degrees, among which Agrin-25, Agrin-344, The coating concentrations of Agrin-530, Agrin-824 and Agrin-1048 were 200ng / ml, 200ng / ml, 250ng / ml, 150ng / ml and 250ng / ml, respectively. Add 100 μL to each well of a 96-well microtiter plate, and set it to adsorb at 2-8°C for 24 hours. Empty the coating solution and wash the plate 3 times with washing solution.

[0083] 2, closed

[0084] (1) Blocking solution for coating: Na 2 HPO 4 ·...

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PUM

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Abstract

The invention provides human Agrin antigen, ELISA detection kit, preparation method and application thereof. The human Agrin antigens include those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 The amino acid sequence also provides seven detection kits containing the above seven human Agrin antigens, and one detection kit simultaneously containing the above seven human Agrin antigens. The human Agrin detection kit detects the Agrin antibody in human serum, has strong specificity, high reaction sensitivity, high throughput and low cost, can diagnose myasthenia gravis, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a human Agrin antigen, a human Agrin antibody detection kit and a preparation method and application thereof. Background technique [0002] Myasthenia gravis (MG) is a common neuromuscular junction (NMJ) disease. In most patients, MG may result from an autoimmune response to acetylcholine receptors (AChRs), and autoantibodies to AChRs can be detected in 80%-85% of MG patients. However, AChR antibodies are not detected in approximately 20% of MG patients. There is evidence that these "seronegative" patients can develop antibodies to proteins that are critical for the formation or maintenance of the NMJ. The aggregated protein Agrin, released from motor neurons, binds to low-density lipoprotein receptor-related protein 4 (LRP4) and activates the receptor tyrosine kinase MuSK to direct NMJ formation. About 40%-70% of seronegative patients have MuSK autoantibodies. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/70G01N33/68G01N33/558
Inventor 张崇珍王颖郝洪军
Owner WUHAN EASYDIAGNOSIS BIOMEDICINE
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