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Detection material, preparation method and application of anti-ampa1 autoantibody in human body fluid

A 17T2A-AMPA1, detection material technology, 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.

Active Publication Date: 2021-03-23
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 purpose of the present invention is to provide a detection material for detecting AMPA1 autoantibodies, a preparation method and application of the material, and solve the problems of time-consuming detection, cumbersome steps, and antibody demand of existing detection methods. Large size, high cost, low sensitivity, etc.

Method used

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  • Detection material, preparation method and application of anti-ampa1 autoantibody in human body fluid
  • Detection material, preparation method and application of anti-ampa1 autoantibody in human body fluid
  • Detection material, preparation method and application of anti-ampa1 autoantibody in human body fluid

Examples

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Embodiment 1

[0038] Step 1, plasmid construction: Obtain the CDS sequence of AMPA1 as the target gene, insert the target gene with restriction sites into the 17T2A plasmid vector to obtain the recombinant plasmid vector 17T2A-AMPA1, and extract the plasmid for subsequent experiments after the sequencing is correct. The plasmid vector construction of example specifically comprises the following steps:

[0039] Step 1.1: Obtain the CDS sequence of AMPA1 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;

[0040] 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-AMPA1;

[0041] 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. The map of...

Embodiment 2

[0065] This embodiment discloses a method for preparing a sealing material, which specifically includes:

[0066] 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;

[0067] 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

[0071] The detection material for anti-AMPA1 autoantibodies in human body fluid prepared in Example 1 is used to detect the AMPA1 antibody in the sample. Specifically, the detection process for detecting the AMPA1 antibody in the sample includes:

[0072] 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.

[0073] 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.

[0074] 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 ...

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Abstract

The invention discloses a detection material of an anti-AMPA1 autoantibody in human body fluid and a preparation method and application of the detection material. An anti-AMPA1 receptor detection material is prepared by the way that an NC membrane is coated with an AMPA1 antigen, and a specific AMPA1 antibody in human serum and cerebrospinal fluid is bound to the antigen, color development is performed by alkaline phosphatase substrate-ligand reaction, a blocking material is added to color development reaction, and whether a detected sample contains the AMPA1 antibody or not can be directly judged by visual observation. The method has the advantages such as high sensitivity, easy operation, and rapid detection, and is helpful to the identification and diagnosis of anti-AMPA1 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-AMPA1 autoantibody in human body fluid. Background technique [0002] α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR) is an ionotropic glutamate receptor Protein family, divided into GluR1 and GluR2 subtypes (known as AMPA1 and AMPA1), mainly expressed in the hippocampal neuropil, mediating rapid excitatory synaptic transmission in the brain. Studies have found that anti-AMPAR antibodies can lead to a decrease in the number of GluR1 / 2 receptors on the presynaptic or post-synaptic membranes of hippocampal neurons, mostly manifesting as symptoms of limbic encephalitis such as memory loss, confusion, and behavioral abnormalities. In 2009, Meizan Lai et al. discovered a new type of autoimmune cell surface antigen -...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C07K1/14C12N15/867G01N33/531G01N33/564
CPCC07K14/70571C12N15/86C12N2740/15043C12N2800/107G01N33/531G01N33/564G01N2333/70571G01N2800/28
Inventor 闫亚平李怡婷李科
Owner SHAANXI MYBIOTECH CO LTD
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