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Antigen protein combination for detection, diagnosis or risk prediction of Alzheimer, and kit containing antigen protein combination

An Alzheimer's disease, antigen protein technology, applied in the field of biological detection, can solve problems such as staying in a small research stage, difficult to detect autoantibodies, lack of large-scale and reproducible clinical data, etc.

Active Publication Date: 2019-05-10
湖南诺琪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to technical limitations, many autoantibodies in Alzheimer's disease are difficult to detect in the early stage; at the same time, although a variety of autoantibodies have been reported to be related to AD, it is not yet known which antibodies have broad clinical significance
Moreover, even if some autoantibodies are considered to be related to AD, they are only in the research stage of a small scope, and there is still a lack of large-scale and reproducible clinical data to prove it.

Method used

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  • Antigen protein combination for detection, diagnosis or risk prediction of Alzheimer, and kit containing antigen protein combination
  • Antigen protein combination for detection, diagnosis or risk prediction of Alzheimer, and kit containing antigen protein combination
  • Antigen protein combination for detection, diagnosis or risk prediction of Alzheimer, and kit containing antigen protein combination

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0070] Preparation method of blocking solution: dissolve 5g of casein in PBS solution, then dilute to 1L;

[0071] Preparation method of 20× washing solution: 20×PBS, pH7.4, add 0.5% Tween20;

[0072] Preparation method of serum / antibody diluent: dissolve 1g casein and 10g bovine serum albumin in PBS solution, then dilute to 1L;

[0073] The preparation method of TMB chromogen: 50mM imidazole buffer pH 5, 7.5mM PEG3350, 2.94mM urea hydrogen peroxide, 1.6mM TMB;

[0074] Preparation method of stop solution: 2M sulfuric acid.

Embodiment 1

[0079] Example 1 Protein Screening for Antigens as Autoantibodies

[0080] Through literature search, 50 known proteins related to Alzheimer's disease were selected, as shown in Table 2.

[0081] Table 2. Sequences of proteins to be tested

[0082]

[0083]

[0084]Using a human cDNA library (purchased from Invitrogen) or DNA synthesized from the whole gene as a template, PCR amplification or enzyme digestion is used to prepare the required recombinant antigenic protein fragments. For example, after the amplified PCR product is purified, it is connected to the pET28 vector, and appropriate tags such as HIS and c-myc are added upstream of the antigen coding sequence to form a fusion protein, and then the recombinant vector is transformed into E. coli DH5α competent cells middle. The obtained recombinant plasmid was identified by colony PCR, enzyme digestion and sequencing to confirm that it contained the correct foreign fragment. Transform the obtained recombinant pl...

Embodiment 2

[0093] Example 2 Detection performance of candidate antigen protein combinations in Alzheimer's disease patients and healthy subjects

[0094] Patients with Alzheimer's disease and healthy subjects were scored according to the Mini-Mental State Examination (Mini-Mental State Examination, MMSE)-Folstein version, and those with a score of 27-30 were normal people, and those with a score of 21-26 Testers with a score of 10-20 are considered moderate (middle stage) patients, and testers with a score of 0-9 are severe (late stage) patients.

[0095] According to the MMSE score, 120 cases of Alzheimer's disease patients in the early stage, 87 cases in the middle stage, and 75 cases in the late stage were taken as test samples, and 94 healthy subjects with normal MMSE scores were taken as samples of healthy subjects to test the antigen proteins of each group Combined Sensitivity and Specificity.

[0096] According to the screening results in Example 1, a variety of proteins were s...

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Abstract

The invention provides a kit for detecting an Alzheimer autoantibody in serum. The kit comprises an antigen protein combination, the antigen protein combination contains at least four proteins, and the proteins are selected from RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8, CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1 and MRPL34. The inventionfurther provides application of the limited antigen protein combination to preparation of a reagent for detection, diagnosis or risk prediction of Alzheimer, and methods for detection, diagnosis or risk prediction of Alzheimer.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular, the invention relates to an antigen combination for detecting autoantibodies in a mammalian sample and application thereof, so as to realize early diagnosis of whether the mammal suffers from Alzheimer's disease. Background technique [0002] Alzheimer's disease (AD), commonly known as senile dementia, is a group of primary degenerative brain degenerative diseases of unknown etiology. The clinical manifestations of Alzheimer's disease are mainly loss of memory and other cognitive functions. The onset is slow and difficult to detect. It is characterized by a pre-symptomatic period that may last for several years. At the same time, patients have developed neurological symptoms before clinical symptoms appear. degenerates and has a rather long course after onset. [0003] According to the World Health Organization, 10% of the elderly over 65 years old in the world today will devel...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 曹利勤孙苏彭
Owner 湖南诺琪生物科技有限公司
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