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Human Agrin antigen and human Agrin antibody detecting kits and preparing method and application thereof

A technology of antibody detection and kit, which is applied in the field of biopharmaceuticals, can solve the problems of not being detected, and achieve the effect of strong specificity, high sensitivity and good stability

Active Publication Date: 2019-05-10
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 and human Agrin antibody detecting kits and preparing method and application thereof
  • Human Agrin antigen and human Agrin antibody detecting kits and preparing method and application thereof
  • Human Agrin antigen and human Agrin antibody detecting kits and preparing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 constructs recombinant expression plasmid and engineering bacterium

[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 the Agrin protein was analyzed, including hydrophilicity and surface accessibility, and combined with its spatial conformation and modification characteristics of each domain, the fragments of the seven regions of the mature Agrin protein were selected to obtain separately. After a series of research, the present inventor 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 gene-synthesized, and...

Embodiment 2

[0066] Expression and purification of embodiment 2 antigen

[0067] 1. Use the constructed recombinant protein expression engineering bacteria to conduct induction expression experiments. Inoculate 7 recombinant bacteria into 600ml LB culture solution (ingredients: 10g sodium chloride / liter, 10g peptone / liter and 5g yeast extract / liter), shake the bacteria at 37 degrees and 200RPM until OD600 reaches 0.6-0.8, press Add 24mg / ml IPTG at 1:1000 for induction for 4 hours. Centrifuge, collect bacteria, and prepare for purification.

[0068] 2. The filler selected for purification is Ni Sepharose (product number: 17-0729-10) from GE, and the following solutions were 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 solutions for purification of inclusion bodie...

Embodiment 3

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

[0079] 1. Coated microtiter plate

[0080] (1) Coating solution: NaCl 8.5g, NaCl 2 HPO 4 12H 2 O 30.8g, KH 2 PO 4 2.2g, add ddH 2 0 to 1000ml, adjust the pH to 7.4.

[0081] (2) Washing solution 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 0 to 1000ml, adjusted to pH7.4.

[0082] (3) Coating method: Coat the 7 Agrin antigen fragments with 0.1M PBS (PH7.4) coating buffer in the wells of the microtiter plate, overnight at 4, among which Agrin-25, Agrin-344, 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 ELISA plate, and place at 2-8°C for 24 hours to absorb. Empty the coating solution, and wash the plate 3 times with the washing solution.

[0083] 2. Closed

[0084] (1) Blocking solution for coatin...

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PUM

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Abstract

The invention provides human Agrin antigen and ELISA detecting kits and a preparing method and application thereof. Human Agrin antigens have the amino acid sequences 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 invention further provides the seven detecting kits with the seven human Agrin antigens respectively and the detecting kit with the seven human Agrin antigens simultaneously. The human Agrin detecting kit detects Agrin antibodies in human serum, is high in specificity, reaction sensitivity and flux and low in cost, can diagnose yasthenia gravis symptoms and is suitable for large-scale application and popularization.

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 muscle nerve junction (neuromuscular junction, NMJ) disease. In most patients, MG may arise 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 critical for NMJ formation or maintenance. The aggregated protein Agrin, released from motor neurons, binds to low-density lipoprotein receptor-related protein 4 (LRP4), activating the receptor tyrosine kinase MuSK to direct NMJ formation. Approximately 40%-70% of seronegative patients have MuSK autoantibodi...

Claims

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Application Information

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Patent Type & Authority Applications(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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