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Protein antigen composition for detecting Alzheimer's disease autoantibody and application of composition

A technology for Alzheimer's disease and antigens, which is applied in the field of protein antigen combination detection of Alzheimer's disease autoantibodies in human serum samples, and can solve problems such as poor detection effect, large number of protein antigens, and misdiagnosis

Active Publication Date: 2020-02-28
SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the number of protein antigens disclosed in CN103154736A is extremely large, and it is still necessary to select proteins that can be used in combination as a combination of protein antigens to detect Alzheimer's autoantibodies that may exist in the subject's serum with high specificity and sensitivity , to provide a practical means of diagnosing or staging Alzheimer's disease
Moreover, even for a single protein antigen, the binding effect between the full length of the protein and the corresponding Alzheimer's autoantibody that may exist may be weak, resulting in poor detection results, resulting in detection failure or even misdiagnosis

Method used

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  • Protein antigen composition for detecting Alzheimer's disease autoantibody and application of composition
  • Protein antigen composition for detecting Alzheimer's disease autoantibody and application of composition
  • Protein antigen composition for detecting Alzheimer's disease autoantibody and application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Recombinant Vector Construction, Expression and Purification of Antigen

[0037] 1. The choice of antigen. 115 antigenic proteins highly related to Alzheimer's disease were selected for construction, expression and purification. In order to save the length of the article, only 50 of them are representative in this article, and their respective database IDs are shown in Table 1.

[0038] Table 1 Database ID correspondence table of proteins to be tested

[0039]

[0040] 2. Construction and expression of antigen recombinant vector. The target protein antigen was screened from the 50 proteins listed in Table 1. Using the human cDNA library (purchased from Invitrogen) or the whole gene synthetic DNA as a template, primers were designed respectively, and the full-length gene of the protein was cloned into the pET28 plasmid by molecular cloning methods such as PCR, enzyme digestion, and ligation. At the same time, tags such as HIS and c-myc are added to the ...

Embodiment 2

[0043] Example 2: Screening and obtaining candidate antigens from proteins to be tested

[0044] 1. The solutions and reagents used in this example are as follows:

[0045] (1) Coating buffer is PBS buffer, pH=7.4. Its preparation method is: accurately weigh 3.58g Na 2 HPO 4 12H 2 O, 0.23g KH 2 PO 4 2H 2 O, 0.2g KCl, 8.0g NaCl, dissolved in water, dilute to 1L with water.

[0046] (2) Blocking Solution / Sample Diluent / Antibody Diluent: Dissolve 10g of BSA (bovine serum albumin) in the coating buffer, and dilute to 1L with water.

[0047] (3) Washing liquid: ready-to-use. Add 0.5% Tween20 (V / V) to the coating buffer before use, pH=7.4.

[0048] (4) TMB color reagent, purchased from KPL company.

[0049] (5) Termination solution: 1M hydrochloric acid.

[0050] 2. The solid phase is coated with the protein to be tested. The purified protein to be tested obtained in Example 1 was diluted to 5 μg / ml with coating buffer, added to a 96-well plate, 50 μl per well, and coate...

Embodiment 3

[0061] Example 3: Active site screening of candidate antigens

[0062] 1. Analyze the amino acid sequence and structure of the candidate antigens obtained in Example 2, and select different sequence fragments and antigenic epitopes from each of the candidate antigens after a large number of preliminary tests. The selected sequence fragments or full lengths are shown in Table 4.

[0063] Table 4 Correspondence table of sequence fragments intercepted on each candidate antigen

[0064]

[0065] 2. According to the method of Example 1, the protein fragments in Table 4 were constructed, expressed and purified as expression vectors to obtain the constructed protein fragments, and then detected according to the method of Example 2 when each constructed protein fragment was used as an antigen. Sensitivity and Specificity. Similar to Example 1, although some tags are added to each protein fragment here, the purpose of adding tags is mainly to facilitate extraction and identificati...

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Abstract

The invention belongs to the field of biological detection, in particular to a protein antigen composition for detecting an Alzheimer's disease autoantibody in a human serum sample and an applicationof the composition. The adopted technical scheme includes that the antigen composition comprises at least two protein fragments of the following proteins: MAPT, ADARB1, HSP60, P21, DAG, DNAJC8, RAGE,ASXL1 and JMJD2D. Based on the above antigen composition, a kit for diagnosis, especially early diagnosis of Alzheimer's disease or related risks can be prepared. By using the antigen composition, early Alzheimer's disease can be diagnosed pertinently, rapidly and accurately, and thus the composition has important practical significance.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a protein antigen combination for detecting autoantibodies of Alzheimer's disease in human serum samples and an application thereof. Background technique [0002] Alzheimer's disease (AD for short) is a progressive neurodegenerative disease mainly characterized by memory and cognitive dysfunction. According to the development of cognitive dysfunction, Alzheimer's disease can be divided into early, middle and late stages. In the early stage, patients have no obvious symptoms, and most of them only show forgetfulness and anxiety, so it is difficult to detect or diagnose; in the middle stage, chaotic symptoms will appear, personality and temper will change, and memory will also appear confused; Dementia, the inability to take care of oneself, eventually leads to death due to multiple diseases. [0003] The current diagnostic methods for Alzheimer's disease include t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N2800/2821G01N2800/52C07K14/47G01N33/6893G01N33/6896G01N33/564G01N2800/54G01N2800/56C07K14/4711C12N9/78C12Y305/04C07K14/4738C07K14/4702C07K14/70503C12N9/0071C12Y114/11027G01N33/532
Inventor 邹永红
Owner SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD
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