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Cell slide for detecting acetylcholine receptor autoantibody in human body fluid as well as preparation method and application of cell slide

An acetylcholine receptor, autoantibody technology, applied in receptors/cell surface antigens/cell surface determinants, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve whether it is necessary or not. Conclusion, inability to distinguish Rapsyn and AchR antibody positive, no immunofluorescence detection kit, etc., to achieve high sensitivity and good application prospects

Active Publication Date: 2021-11-16
SHAANXI MYBIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. The existing research results have no clear conclusion on whether the AchRα subunit P3A sequence is necessary in the detection of positive samples;
[0007] 2. Rapsyn is a kind of self-antigen. It acts as an auxiliary protein to promote the correct folding of AchR subunits. Transiently transfect it with AchR subunits to obtain antigen cells that overexpress AchR. Rapsyn antigens will be expressed. When the sample is tested, it may not be possible to distinguish between Rapsyn and AchR antibody positive, resulting in false positive results;
[0008] 3. Immunofluorescence is an available auxiliary detection method, and there is no commercialized immunofluorescence detection kit at present

Method used

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  • Cell slide for detecting acetylcholine receptor autoantibody in human body fluid as well as preparation method and application of cell slide
  • Cell slide for detecting acetylcholine receptor autoantibody in human body fluid as well as preparation method and application of cell slide
  • Cell slide for detecting acetylcholine receptor autoantibody in human body fluid as well as preparation method and application of cell slide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Step 1, construction of recombinant vector

[0034] By PCR or artificial synthesis method, each subunit gene of AchR α1(P3A+), α1(P3A-), β1, δ1, δ2, δ3, δ4, ε, γ and Rapsyn(longer), (wherein, α1( P3A+), α1 (P3A-), δ1, δ2, δ3, δ4 sequences such as SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, Shown in SEQ.ID.NO.5 and SEQ.ID.NO.6, the sequence numbers of other sequences on NCBI are: NM_000747.3, NM_000080.4, NM_005199.5, NM_005055.5) connected by molecular cloning On pCDNA3.1, the constructed recombinant vector is sequenced correctly and extracted for later use;

[0035] Step 2. Cell transfection

[0036] (1) 293T cell culture: 10% FBS-DMEM high-glucose medium was prepared by DMEM high-glucose medium and FBS at a ratio of 9:1, and passaged at a ratio of 1:5 to 1:6 when the cells were confluent, placed at 37°C, Cultivate overnight in a cell culture incubator with 5% CO2;

[0037] (2) When the cell density is 30% to 40%, transfect each gene according to the combin...

Embodiment 2

[0058] 1. Construction of recombinant vector

[0059] By PCR or artificial synthesis, connect Rapsyn (shorter, the sequence number on NCBI is: NM_032645.5), and the gene is connected to pCDNA3.1 by molecular cloning method, and the rest of the required vectors are all constructed in Example 1 Vector, the constructed recombinant vector is sequenced correctly and extracted for later use;

[0060] 2. Cell transfection

[0061] (1) 293T cell culture: 10% FBS-DMEM high-glucose medium was prepared by DMEM high-glucose medium and FBS at a ratio of 9:1, and passaged at a ratio of 1:5 to 1:6 when the cells were confluent, placed at 37°C, Cultivate overnight in a cell culture incubator with 5% CO2;

[0062] (2) When the cell density is 30% to 40%, transfect each gene according to the combination of Table 2 and Table 3:

[0063] Transfection System 1 (Table 1):

[0064] Get the recombinant vectors pCDNA3.1-α1 (P3A-), pCDNA3.1-β1, pCDNA3.1-δ1, pCDNA3.1-ε, pCDNA3.1-γ, pCDNA3. 1-rapsyn...

Embodiment 3

[0104] 1. 293T cell culture: 10% FBS-DMEM high-glucose medium was prepared by DMEM high-glucose medium and FBS at a ratio of 9:1, and passaged at a ratio of 1:5 to 1:6 when the cells were confluent, and placed at 37°C for 5 Culture overnight in a cell culture incubator with %CO2;

[0105] 2. When the cell density is 30%-40%, take the vector constructed in Example 1 and carry out transfection according to the combination of the following three methods, replace with fresh culture medium after 4 hours, and fix the cells after 48 hours.

[0106] (1) Adult + fetal type: pCDNA3.1-α1(P3A-), pCDNA3.1-β1, pCDNA3.1-γ, pCDNA3.1-δ1, pCDNA3.1-ε, 17-T2A, plasmids according to 2 :1:1:1:1:24 ratio for transfection, the total amount of transfection plasmid: 6ug;

[0107] (2) Adult type: pCDNA3.1-α1(P3A-), pCDNA3.1-β1, pCDNA3.1-γ, pCDNA3.1-δ1, 17-T2A, plasmids according to 2:1:1:1:24 Ratio for transfection, the total amount of transfected plasmids: 6ug;

[0108] (3) Fetal type: pCDNA3.1-α1(P...

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Abstract

The invention discloses a cell slide for detecting an acetylcholine receptor autoantibody in human body fluid as well as a preparation method and application of the cell slide, and belongs to the technical field of biology. According to the scheme, the method comprises the following steps: co-transfecting each subunit of an acetylcholine receptor and a no-load plasmid according to a certain proportion, and then fixing, washing, terminating, washing and drying to prepare the cell slide for detecting the autoantibody of the acetylcholine receptor in human body fluid. Compared with a radioimmunoassay and an enzyme-linked immunosorbent assay, the method has higher sensitivity; compared with an immunofluorescence method reported in literatures in the prior art, the method has the advantage that the detection specificity is improved while the background is reduced. Therefore, from the perspective of research or as an auxiliary diagnosis means of the myasthenia gravis disease, the method has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a cell slide for detecting acetylcholine receptor autoantibodies, a preparation method and application thereof. Background technique [0002] Myasthenia gravis (MG) is an autoimmune disease with acetylcholine receptor antibody (AchR-Ab)-mediated transmission disorder at the nerve-muscle junction (NMJ), and the lesion mainly involves acetylcholine receptors on the postsynaptic membrane of NMJ (acetyl cholinergic receptor, AchR). At present, the methods for detecting autoantibodies in myasthenia gravis mainly include radioimmunoassay and enzyme-linked immunoassay. There is now evidence that some samples from patients with symptoms of myasthenia gravis are negative by radioimmunoassay, and the enzyme-linked immunosorbent assay has the same problem as radioimmunoassay. Cell-based immunofluorescence (CBA, Cell Based Assay) overcomes the shortcomings of the above two methods. By expressing...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/85C12N5/10G01N33/68G01N33/543
CPCC07K14/70571C12N15/85G01N33/6893G01N33/6854G01N33/54306C12N2800/107G01N2333/70571G01N2800/10
Inventor 闫亚平赵子越郝文斌封雪李科
Owner SHAANXI MYBIOTECH CO LTD
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