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
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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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