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Detection material for resisting CASPR2 autoantibodies in human body fluid, preparation method and application

A 17T2A-CASPR2 and autoantibody technology, applied in the field of biomedicine, can solve the problems of large antibody demand, long detection time, cumbersome steps, etc., and achieve the effect of strong operation skills, short detection cycle and high detection cost

Active 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

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a detection material for detecting CASPR2 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 for resisting CASPR2 autoantibodies in human body fluid, preparation method and application
  • Detection material for resisting CASPR2 autoantibodies in human body fluid, preparation method and application
  • Detection material for resisting CASPR2 autoantibodies in human body fluid, preparation method and application

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Embodiment 1

[0037] Step 1, plasmid construction: obtain the CDS sequence of CASPR2 as the target gene, insert the target gene with restriction sites into the 17T2A plasmid vector to obtain the recombinant plasmid vector 17T2A-CASPR2, and extract the plasmid for subsequent experiments after the sequencing is correct. The plasmid vector construction of example specifically comprises the following steps:

[0038] Step 1.1: Obtain the CDS sequence of CASPR2 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;

[0039] 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-CASPR2;

[0040] 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 ...

Embodiment 2

[0064] This embodiment discloses a method for preparing a sealing material, which specifically includes:

[0065] 1) Cut the carrier film (glass fiber mat in this example) into small square pieces of 0.5cm×0.5cm, put 15 μL of 2.0M sucrose solution on each small piece of glass fiber mat, bake at 100°C for 20min, and store at room temperature for later use;

[0066] 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 , vortexed for a few sec...

Embodiment 3

[0070] The detection material for anti-CASPR2 autoantibodies in human body fluid prepared in Example 1 is used to detect CASPR2 antibodies in samples. Specifically, the detection process for detecting CASPR2 antibodies in samples includes:

[0071] 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.

[0072] 2) Serum blocking: the sample to be tested was diluted with working solution (1×PBS, 0.5% Triton X-100, 0.04% EDTA) at a ratio of 1:250, and a piece of blocking material prepared in Example 4 was added to every 250 μL of diluted serum. After standing at room temperature for 2 minutes, vortex for a few seconds, and then stand at room temperature for 5 minutes.

[0073] 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 ...

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Abstract

The invention discloses a detection material for resisting CASPR2 autoantibodies in human body fluid, a preparation method and an application. A CASPR2 antigen is arranged on an NC membrane through coating, a detection material for resisting a CASPR2 receptor is prepared, a specific CASPR2 antibody in human serum and cerebrospinal fluid can be combined with the antigen, color development is performed through an alkaline phosphatase substrate-ligand reaction, in a color development reaction, an enclosing material disclosed by the invention is added, and whether CASPR2 antibodies are contained in detection samples can be directly judged through observation by naked eye. The method has the advantages of being high in sensitivity, simple and convenient to operate, quick to detect and the like,and recognition and diagnosis of CASPR2 receptor resistant encephalitis are facilitated.

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-CASPR2 autoantibody in human body fluid. Background technique [0002] Contactin Associated Protein Like 2 (CASPR2) belongs to the family of axonal proteins that mediate the interaction between neurons and glia during nervous system development and are also involved in the differentiation of intraaxonal potassium The localization of ion voltage-gated potassium channel (Voltage-gated potassium channel, VGKC). The protein has been implicated in a variety of neurodevelopmental disorders, including Morvan syndrome, schizophrenia, epilepsy, autism, intellectual disability and autoimmune encephalitis. In 2010, Irani and Lai et al. found that the disease previously known as VGKC encephalitis, the actual pathogenic antibody is against CASPR2, not VGKC itself, and named CASPR2 antibody autopositive encephali...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K1/14C12N15/867G01N33/531G01N33/564
CPCC07K14/70571C12N15/86C12N2740/15043C12N2800/107G01N33/531G01N33/564G01N2333/70571G01N2800/28
Inventor 闫亚平李怡婷冯昆李科
Owner SHAANXI MYBIOTECH CO LTD
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