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Application of anti-CAMK2A autoantibody reagent in preparation of kit for diagnosing nervous system symptom related diseases

An autoantibody, nervous system technology, applied in the fields of resistance to vector-borne diseases, disease diagnosis, biochemical equipment and methods, etc.

Active Publication Date: 2022-02-08
SHAANXI MYBIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, although progress has been made in identifying autoantibody-mediated immune mechanisms in patients with autoimmune encephalitis, there are still a proportion of patients who exhibit encephalitic symptoms with negative serological test results. Therefore, identification of autoantibody-binding Neoantigens, improving existing diagnostics and therapeutics, remain a daunting task

Method used

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  • Application of anti-CAMK2A autoantibody reagent in preparation of kit for diagnosing nervous system symptom related diseases
  • Application of anti-CAMK2A autoantibody reagent in preparation of kit for diagnosing nervous system symptom related diseases
  • Application of anti-CAMK2A autoantibody reagent in preparation of kit for diagnosing nervous system symptom related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Rat primary neurons immunofluorescence

[0066] Step 1 rat primary neurons isolated

[0067] 1,10% chloral hydrate anesthesia pregnant rats, 75% ethanol immersion disinfection 3min, biological safety cabinet fetuses removed;

[0068] 2, using a cold HBSS (137mM NaCl, 5.4mM KCl, 0.6mM MgSO 4 · 7h 2 O, 0.5mM MgCl 2 · 6h 2 O, 0.3mM Na 2 HPO 4 · 2h 2 O, 0.4mM kH 2 PO 4 , 5.6mM Glucose, 4.2mM NaHCO 3 , PH7.2 ~ 7.4) fetal rinsed twice with pre-cooled dissected out in HBSS soaked in fetal brain pre-cooled in DMEM;

[0069] 3, disposable DMEM, with ophthalmic scissors to cut tissue pieces 1m 3 Minced mixtures size. Papain was added (at a concentration of 2mg / ml). In 10cm dishes at 37 ℃ for 30 minutes, gently shaking every 5min. Usually 10-15 Brain tissue with digestive 20ml;

[0070] 4, after digestion, the cells were moved to a 50ml centrifuge tube, resuspended 20ml DMEM was added, 1ml pipetting tip 10, 400g, 4 ℃ centrifuge 5min,

[0071] 5, the supernatant was discarded was add...

Embodiment 2

[0096] Immunoprecipitation of Camk2a

[0097] There are three serums used in the immunoprecipitation experiment of the present embodiment, which are serum, healthy subject 1 and a healthy subject 2 of the patient 1, respectively, together with three eluent. The specific experimental steps are as follows:

[0098] 1. Take a 6-dish rat primary neuron cell, abandoned, PBS washed 2 times, 0.4% polymethylene formaldehyde fixed 10 min, discarded, 1 × hepes washed 3 times;

[0099] 2, serum incubation: 10 ul of serum is added to 10 ml of DMEM (1: 100 dilution), and the filtrate is added to a fixed cell with a 0.22 um filter, and the filtrate is incubated for 2 h.

[0100] 3, take 15 ul GE's Proteina to 2ML centrifuge tube, balance liquid (20mm Na 2 HPO 4 , 150 mMNACL was washed 3 times, and the 2H was closed with 300 ul 4% BSA;

[0101] 4, the cells will be placed on ice, abandoned, PBS washed 2 times, add 500 ul lysate (150 mM NaCl, 1 mM EDTA, 100 mM Tris-HCl, 0.5% deoxycholic acid, 1% ...

Embodiment 3

[0109] Immunohombo

[0110] 1. Electrophoresis: Take the sample of the sample after the treatment of the sample for SDS-PAGE;

[0111] 2, the film: After the electrophoresis is completed, the wet film is used, the film film is 200mA, 80 min;

[0112] 3, closed: 5% degreasing milk powder room temperature is closed for 1 h;

[0113] 4, an anti-incubation: incubation anti-Camk2a polyclonal antibody (antibody purchased from Sigma) according to the proportion of 1: 2000, and incubated for 2 h room temperature;

[0114] 5, wash: TBST wash 3 times, 5 min each time;

[0115] 6, two anti-incubation: adding the second anti-HRP mark, incubation for 1 h room temperature

[0116] 7. Wash the TBST 3 times, 5 min each time;

[0117] 8, color color: Add chemiluminum, record results.

[0118] Experimental results:

[0119] image 3 The wb identification results showing the serum immunoprecipitate, wherein M: Marker; 1: The sample is a serum immunopropiator in patients; 2: Sample is a healthy subje...

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PUM

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Abstract

The invention relates to application of an anti-CAMK2A autoantibody reagent in preparation of a kit for diagnosing nervous system symptom related diseases, and belongs to the technical field of disease diagnosis kits. The invention provides an application of an anti-CAMK2A autoantibody reagent in preparation of a kit for diagnosing diseases related to nervous system symptoms. The kit prepared by the application can realize the detection of the CAMK2A autoantibody, and can be used for diagnosing nervous system diseases, especially nervous system diseases related to one or more symptoms; wherein the symptoms comprise sleep disorder, sleepiness, depression, cognitive and mental disorder, autonomic nerve dysfunction, epileptic seizure, consciousness level decline and consciousness blurring. The kit can also be used for distinguishing autoimmune diseases, especially autoimmune diseases and non-autoimmune diseases of a nervous system.

Description

Technical field [0001] The present invention relates to the field of disease diagnostic kit technology, and more particularly to the application of reagents for anti-CAMK2A autoantibodies in the preparation of diagnostic neurological symptoms related diseases. Background technique [0002] Autoimmune Encephalitis, AE, is a class of encephalitis mediated by autoimmune mechanisms, is a central nervous system of antibody-mediated damage and a central nervous system of neuronal structural dysfunction, including antibodies comprising Anti-neuronal intracellular antigenic antibody (a sub-tumor syndrome) and antibiotic cell surface antigen antibody. [0003] Existing literature reports and clinically detected autoimmune encephalitis antibody spectroscopy include: N-methyl-D aspartic acid receptor (NMDAR), α-amino-3 hydroxy-5 methyl-4 isoxazole Receptor (ampar), luminine-rich glutamic neuroprotalaximin 1 (LGI1), contact protein-related protein-2 (CasPR2), γ-amino acid receptor B (gabaBAB...

Claims

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Application Information

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IPC IPC(8): G01N33/573C12N9/12
CPCG01N33/573C12N9/1205G01N2800/28G01N2800/24G01N2333/9121Y02A50/30
Inventor 闫亚平任妮柴单单曹玉妍封雪李科
Owner SHAANXI MYBIOTECH CO LTD
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