Autoimmune encephalitis antibody transient transfection and stable transfection detection method and application thereof

A technology of antibodies and transfection reagents, applied in the medical field, can solve problems such as poor repeatability, inaccurate measurement, and cell death

Pending Publication Date: 2021-12-07
陈向军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has disadvantages, such as NMDAR subunits (NR1 and NR2A), causing massive cell death, resulting in inaccurate and poorly reproducible measurements

Method used

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  • Autoimmune encephalitis antibody transient transfection and stable transfection detection method and application thereof
  • Autoimmune encephalitis antibody transient transfection and stable transfection detection method and application thereof
  • Autoimmune encephalitis antibody transient transfection and stable transfection detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] Example 1 Establishment of autoantibody detection method in AE based on transiently transfected cells

[0166] 1.1 Group transfection of HEK293 cells:

[0167] (1) Group N1 / 2A: wild-type GRIN1 plasmid (article number: P6908, company: Miaoling Plasmid Platform) + GRIN2A plasmid (article number: P8212, company: Miaoling Plasmid Platform).

[0168] (2) N1-815R / 2A group: pCDNA3.1-GRIN1-815R-GFP plasmid + GRIN2A plasmid (product number: P8212, company: Miaoling plasmid platform). Among them, the purchased human wild-type pcDNA 3.1-GRIN1 plasmid (article number: P6908, company: Miaoling Plasmid Platform) was gene-edited and nucleotide mutations were introduced to mutate the 815th amino acid from glycine to arginine, and then A GFP tag (fused to express green fluorescent protein) was added to the plasmid to obtain the plasmid pCDNA3.1-GRIN1-815R-GFP.

[0169] (3) blank control;

[0170] 4 µg of total DNA in a 35 mm cell culture well was used to transfect cells. In cells tr...

Embodiment 2

[0181] Example 2: Establishment of autoantibody detection method in AE based on transiently transfected cells

[0182] The purpose of this example: to establish an efficient and reliable system for the detection of common autoantibodies in AE based on the indirect immunofluorescence method of transiently transfected HEK293 cells. Methods as below:

[0183] Use the existing commercial cDNA library to obtain 6 common autoantibodies (NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R) Corresponding target gene. The 815th amino acid of NMDAR (GluN1 protein) was mutated from glycine to arginine, LGI1 was fused to express the transmembrane region of Caspr2, and the genes of other autoantibody proteins remained unchanged. The cDNA of these autoantibodies was then edited such that they were fused to a gene fragment expressing green fluorescent protein (group transfection).

[0184] HEK293 cells were inoculated in 96-well plates, and HEK293 cells were grouped and transfected by PEI metho...

Embodiment 3

[0193] Example 3: Exploration of Autoantibody Detection Method in AE Based on Stably Transfected Cells

[0194] Purpose of this example: To produce stably transfected cells as cell substrates for autoantibody detection in AE.

[0195] method:

[0196] The tool plasmid of the PiggyBac transposition system for tetracycline-induced expression is obtained by constructing two inverted repeat sequences in the PiggyBac transposition system to both sides of the sequence required for the tetracycline-induced expression system. The LGI1 (fused to express green fluorescent protein) gene was cloned into the obtained tool plasmid, and after the cells were transfected, 0 μg / ml, 1 μg / ml, and 2 μg / ml doxycycline were added sequentially to verify the tetracycline-induced expression system. And use different concentrations of puromycin and G418 to screen HEK293, and determine the lowest concentration that can kill all cells within 6-8 days as the screening concentration. After screening with ...

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Abstract

The invention provides an autoimmune encephalitis antibody transient transfection and stable transfection detection method and application thereof. Specifically, the invention provides a recombinant cell for expressing mutant NMDAR, the recombinant cell expresses fusion protein of exogenous mutant NMDAR and fluorescent protein, and the transfection survival rate is high. The recombinant cell provided by the invention can be used for detecting antibodies related to autoimmune encephalitis, including NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2 and GABABR. The invention also provides a method for detecting anti-autoimmune encephalitis by using the transfected cell line through an indirect immunofluorescence method based on the recombinant transfected cell line provided by the invention. The method disclosed by the invention has high sensitivity and specificity, and can be used for accurately detecting the types of pathogenic antibodies so as to help a patient to carry out targeted immunotherapy and recover health.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a method for detecting the transient and stable transition of autoimmune encephalitis antibodies and its application. Background technique [0002] Autoimmune encephalitis (Autoimmune encephalitis, AE) is a new type of central nervous system autoimmune disease that has been recognized in the past 10 years. Antibodies, these autoantibodies are considered pathogenic and can be markers for disease diagnosis. AE patients often show severe clinical symptoms such as abnormal mental behavior, epileptic seizures, memory impairment, cognitive abnormalities, motor abnormalities, autonomic dysfunction, and decreased consciousness, but early immunotherapy can achieve good curative effect and disease outcome . In general, common specific autoantibodies that mediate AEs include NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B There are six kinds of R, and the patients with AEs mediated by different antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/62C12N15/12G01N33/68C12R1/91
CPCC12N15/85C07K14/70571G01N33/6896C07K2319/60G01N2333/70571
Inventor 陈向军董强邱玥刘小妮邓波
Owner 陈向军
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