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Detection material for anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody in human body fluid, preparation method and application

A technology of autoantibodies and detection materials, applied in the field of biomedicine, can solve the problems of large demand for antibodies, long detection time, low sensitivity, etc., and achieve the effect of strong operation skills, short detection cycle and high detection cost

Pending Publication Date: 2019-12-24
SHAANXI MYBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide a detection material for detecting MOG autoantibodies, a preparation method and application of the material, and solve the problems of time-consuming detection, cumbersome steps, and antibody demand of the existing detection methods. Large size, high cost, low sensitivity, etc.

Method used

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  • Detection material for anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody in human body fluid, preparation method and application
  • Detection material for anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody in human body fluid, preparation method and application
  • Detection material for anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody in human body fluid, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Step 1, plasmid construction: Obtain the CDS sequence of MOG as the target gene, insert the target gene with restriction sites into the 17T2A plasmid vector to obtain the recombinant plasmid vector 17T2A-MOG, extract the plasmid for subsequent experiments after the sequencing is correct, this implementation The plasmid vector construction of example specifically comprises the following steps:

[0042] Step 1.1: Obtain the CDS sequence of MOG as the target gene by PCR method (artificial synthesis method is also optional), and add SalI / NotI restriction sites at both ends of the target gene;

[0043] Step 1.2: Insert the target gene with the restriction site into the 17T2A plasmid vector, the insertion site is SalI / NotI, to obtain the recombinant vector, which is named 17T2A-MOG;

[0044] Among them, the 17T2A plasmid vector deletes the copGFP element on the basis of the pCDH-CMV-MCS-EF1-copGFP vector, and replaces the FE1 promoter with the T2A element at the same time. Th...

Embodiment 2

[0075] This embodiment discloses a method for preparing a sealing material, which specifically includes:

[0076] 1) Cut the carrier film (glass fiber mat in this example) into small square pieces of 0.5cm×0.5cm, put 15μL of a mixture of 1.9M sucrose and 1.9M glucose on each small piece of glass fiber mat, and bake at 100°C for 20min. Store at room temperature for later use;

[0077] 2) Extraction of 17T2A cell (PS) protein: scrape the 17T2A cells obtained in step 2.3 of the above example with a scraper, collect the 17T2A cells in a 15ml centrifuge tube, 1000rpm, 5min, room temperature, discard the supernatant. Add 1mL of 1×PBS (or normal saline) to the precipitate, pipette to mix, transfer the liquid to a 1.5ml EP tube, 700g, 5min, room temperature, discard the supernatant. Add 1 / 2 volume of the cell pellet volume blocking protein extract (1.25% sodium deoxycholate, 0.25% Triton X-100, 0.75% CHAPS, 20mMNaCl, 2×PI), pipette to mix and transfer the liquid to a 2ml EP tube , v...

Embodiment 3

[0081] The detection material for anti-MOG autoantibodies in human body fluid prepared in Example 1 is used to detect MOG antibodies in samples. Specifically, the detection process for detecting MOG antibodies in samples includes:

[0082] 1) Place the membrane protein-cell membrane complex detection material obtained in step 4 of the above example in a 24-well plate, with the membrane protein antigen-coated side facing up.

[0083] 2) Serum blocking: Dilute the sample to be tested at 1:250 with working solution (1×PBS, 0.5% TritonX-100, 0.04% EDTA), add a piece of blocking material prepared in Example 4 to every 250 μL of diluted serum, and keep at room temperature After standing for 2 minutes, vortex for a few seconds, and then stand at room temperature for 5 minutes.

[0084] 3) Serum incubation: Add the blocked serum to a 24-well plate containing membrane protein-cell membrane complex detection material, 250 μL / well, place the 24-well plate on a horizontal shaker (frequenc...

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Abstract

The invention discloses a detection material for an anti-myelin oligodendrocyte glycoprotein (MOG) autoantibody in human body fluid, a preparation method and application. An MOG antigen coats an NC membrane to prepare the anti-MOG receptor detection material, specific MOG antibodies in human serum and cerebrospinal fluid are combined with the antigen, color development is conducted through an alkaline phosphatase substrate-ligand reaction, a sealing material is added in a color development reaction, and whether a detection sample contains the MOG antibodies or not can be directly judged through visual inspection. The method has the advantages of high sensitivity, easy and convenient operation, quick detection and the like, and is conducive to identification and diagnosis of anti-MOG receptor encephalitis.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a detection material, preparation method and application of anti-MOG autoantibody in human body fluid. Background technique [0002] Antimyelin oligodendrocyte glycoprotein (MOG) encephalitis is an autoimmune disease of the nervous system. According to the International Diagnosis Consensus on MOG Encephalitis (International Consensus for short), evidence based on immunology, neuropathology, serology and population studies shows that MOG encephalitis is different from classic MS and AQP4-IgG positive neuromyelitis optica spectrum disease ( NMOSD), an independent disease entity, is now called MOG-IgG-associated encephalomyelitis (MOG-EM). Among the existing serum MOG-IgG detection methods, the enzyme-linked immunosorbent assay (ELISA) using linearized or denatured MOG peptides as antigens was used to detect MOG-IgG in serum, and the results showed that MOG antibodi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/14C12N15/867G01N33/531G01N33/564
CPCC07K14/4713C12N15/86C12N2740/15043C12N2800/107G01N33/531G01N33/564G01N2333/47G01N2800/28
Inventor 闫亚平黎培李科
Owner SHAANXI MYBIOTECH CO LTD
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