WO2024114615A1 - 含有serf2的蛋白抗原组合及其应用 - Google Patents

含有serf2的蛋白抗原组合及其应用 Download PDF

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WO2024114615A1
WO2024114615A1 PCT/CN2023/134613 CN2023134613W WO2024114615A1 WO 2024114615 A1 WO2024114615 A1 WO 2024114615A1 CN 2023134613 W CN2023134613 W CN 2023134613W WO 2024114615 A1 WO2024114615 A1 WO 2024114615A1
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protein
protein fragment
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amino acid
acid sequence
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PCT/CN2023/134613
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曹利勤
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上海众启生物科技有限公司
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  • the present invention belongs to the field of biological detection, and specifically relates to a SERF2 protein antigen combination and application thereof for distinguishing Alzheimer's disease from frontotemporal dementia and Lewy body dementia and detecting autoantibodies.
  • AD Alzheimer's disease
  • Autoantibodies are antibodies produced by an individual's immune system against an individual's own protein antigens. Under normal circumstances, the immune system produces antibodies in response to exogenous proteins or substances in the body, but sometimes it also recognizes one or more endogenous components of the body, leading to the production of autoantibodies. There is a lot of evidence that the presence of a variety of autoantibodies in serum is involved in neurological diseases and syndromes. We have published a series of antigenic proteins (and/or fragments) and their combinations that can be used for the detection of autoantibodies in AD patients in China Patent CN 110850104 B. These combinations can achieve good results in the clinical diagnosis of Alzheimer's disease (AD), but have not yet involved the differential diagnosis of AD and related dementias such as dementia with Lewy bodies (DLB) and frontal temporal dementia (FTD).
  • AD Alzheimer's disease
  • DLB dementia with Lewy bodies
  • FTD frontal temporal dementia
  • DLB Lewy bodies
  • LB Lewy bodies
  • the two diseases are both disorders, and it is difficult to distinguish them in terms of severity; the content of Tau protein and beta-amyloid protein in the brain and spine is detected, and both have similar changes. Therefore, it is extremely difficult to distinguish the two diseases in clinical practice.
  • clinical differentiation is mainly carried out through imaging examinations.
  • One is through cranial MRI examination, which shows subtle differences in the areas of brain atrophy between the two; the other is through cerebral blood flow SPECT/PET examination, which distinguishes AD and DLB through imaging changes.
  • the existing differentiation methods all require a high level of experience from doctors.
  • Frontotemporal dementia is a progressive neurodegenerative disease with an insidious onset. It is a dementia syndrome characterized by frontotemporal lobe atrophy, accounting for about 5% to 15% of all dementia types. Clinically, both FTD and AD patients have varying degrees of cognitive dysfunction, behavioral disorders, and language disorders, and there is no significant difference in the MMSE scores of FTD patients and AD patients.
  • FTD and AD are generally differentiated by CT and MRI. AD can show widespread brain atrophy, while frontotemporal dementia shows atrophy of the frontal and/or temporal lobes; however, further histopathological examination is still required.
  • Paterson et al. (Alzheimer's Research & Therapy (2016) 10:32) detected the content of 10 proteins, including Tau protein, different fragments of amyloid beta (A ⁇ ) and its precursor protein (APP), NFL and YKL-40, in the cerebrospinal fluid of patients with various dementias, as well as the ratio of related protein content.
  • the results showed that these markers could not effectively distinguish AD from DLB; among them, A ⁇ 42/40 had a certain reference value for distinguishing AD from behavioral variant frontotemporal dementia (bvFTD).
  • bvFTD behavioral variant frontotemporal dementia
  • its disadvantage is that the use of cerebrospinal fluid samples has certain risks for patients and is usually rejected by patients.
  • AD Alzheimer's disease
  • AD is a progressive neurodegenerative disease.
  • the symptoms gradually worsen with the course of the disease.
  • the course of the disease is long and the progression is slow in the early stages of the disease. Therefore, the earlier the diagnosis and intervention, the easier it is to control the disease and the more likely the patient will benefit.
  • the earlier the disease is the lower the accuracy of detection by instruments such as MRI and PET is, because the pathological changes are not obvious.
  • the clinical symptoms of AD are very similar to those of dementias such as FTD and DLB, which further increases the difficulty of clinical diagnosis.
  • the purpose of the present invention is to provide a protein antigen combination and application thereof that can effectively distinguish and identify AD from dementias such as FTD and DLB, and detect autoantibodies.
  • an antigen combination which at least includes SERF2 protein and/or SNAP25 protein.
  • amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7
  • amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8.
  • SERF2 protein and SNAP25 protein are included at the same time.
  • it also includes any one or more of MAPT protein fragments, RAGE protein fragments, ASXL1 protein fragments, JMJD2D protein fragments, P21 protein fragments, and DNAJC8 protein fragments.
  • the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1
  • the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4
  • the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5
  • the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6
  • the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2
  • the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.
  • the antigen combination is used in the preparation of products for detecting/identifying Alzheimer's disease.
  • the antigen combination is used in the preparation of products for distinguishing Alzheimer's disease, frontotemporal dementia, and Lewy body dementia.
  • a kit for detecting Alzheimer's disease is prepared using the antigen combination.
  • SERF2 protein and/or SNAP25 protein and/or MAPT protein fragment and/or RAGE protein fragment and/or ASXL1 protein fragment and/or JMJD2D protein fragment and/or P21 protein fragment and/or DNAJC8 protein fragment in the preparation of a product for detecting/identifying Alzheimer's disease
  • the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7; the SNAP25
  • the amino acid sequence of the protein is shown in SEQ ID NO: 8;
  • the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1;
  • the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4
  • the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5
  • amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6
  • the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2
  • the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.
  • SERF2 protein and/or SNAP25 protein and/or MAPT protein fragment and/or RAGE protein fragment and/or ASXL1 protein fragment and/or JMJD2D protein fragment and/or P21 protein fragment and/or DNAJC8 protein fragment in the preparation of products for distinguishing Alzheimer's disease, frontotemporal dementia and Lewy body dementia
  • the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7
  • the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8
  • the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1
  • the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4
  • the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5
  • amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6
  • the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2
  • the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ
  • the present invention has the following beneficial effects:
  • the present invention provides a new protein antigen composition, which can not only effectively identify AD patients, but also effectively distinguish AD, FTD and DLB, and realize accurate identification of AD, which has important application value and research value.
  • the present application is further explained below in conjunction with specific examples.
  • the experimental methods used in the following examples are all conventional methods; the materials and reagents used can be obtained from commercial sources; the data obtained are the average values obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.
  • Example 1 Construction, expression and purification of recombinant vectors of antigens
  • the recombinant plasmid containing the protein gene fragments in Table 1 was transformed into Escherichia coli BL21 (DE3) competent cells, and clones were picked and inoculated into LB medium and cultured in a shaking incubator at 37°C.
  • the temperature was lowered to 16°C, 0.1 mM isopropylthio- ⁇ -D-galactoside (IPTG) was added to each LB medium, and expression was induced overnight to obtain bacterial cells.
  • Example 2 Screening candidate antigens from the protein to be tested
  • the preparation method is as follows: accurately weigh 3.58 g Na 2 HPO 4 ⁇ 12H 2 O, 0.23 g KH 2 PO 4 ⁇ 2H 2 O, 0.2 g KCl, and 8.0 g NaCl, dissolve them in water, and dilute to 1 L with clean water.
  • Blocking solution/sample diluent/antibody diluent Dissolve 10 g BSA (bovine serum albumin) in coating buffer and dilute to 1 L with water.
  • TMB colorimetric reagent purchased from KPL Company.
  • Solid-phase coating of the test protein Dilute the purified test protein obtained in Example 1 to 5 ⁇ g/ml with coating buffer, add to a 96-well plate, 50 ⁇ l per well, and coat overnight at 4°C. Pour out the solution the next day, spin dry, and wash three times with washing solution, 200 ⁇ l per well each time. Then add 200 ⁇ l of blocking solution to each well, incubate at room temperature for 1 hour, pour out the blocking solution, spin dry, and wash three times with washing solution, 200 ⁇ l per well each time, and spin dry again; obtain the solid-phase coated antigen in the 96-well plate.
  • step 4 add 50 ⁇ l TMB color developer to each well of the 96-well plate, shake for 15 seconds, react in the dark at room temperature for 15 minutes, and then add 50 ⁇ l stop solution; then use an ELISA reader to read the absorbance value at a wavelength of 450 nm to obtain the detection signal (S) of each sample to be tested.
  • Specificity and sensitivity were calculated based on the positive and negative results of the samples. Specificity refers to the proportion of samples from healthy subjects that were correctly judged as negative, that is, the number of samples that were correctly judged as negative in the negative samples divided by the total number of negative samples. Sensitivity refers to the proportion of samples from Alzheimer's patients that were correctly judged as positive, that is, the number of samples that were judged as positive in the positive samples divided by the total number of positive samples. The sensitivity and specificity of sample testing using each tested protein as an antigen were calculated, and the results are shown in Table 2.
  • Example 3 Demonstration of the effect of candidate antigen detection on DLB and FTD patient samples
  • Specificity and sensitivity were calculated based on the positive and negative results of the samples. Specificity refers to the proportion of healthy subject samples correctly judged as negative, that is, the number of negative samples correctly judged as negative divided by the total number of negative samples.
  • DLB sample sensitivity refers to the proportion of DLB patient samples correctly judged as positive, that is, the number of DLB positive samples correctly judged as positive divided by the total number of positive samples.
  • FTD sample sensitivity refers to the proportion of FTD patient samples correctly judged as positive.
  • the proportion of positive samples is the number of positive samples divided by the total number of positive samples in the FTD positive samples.
  • Example 4 Demonstration of the effect of candidate antigen combination in detecting AD, FTD and DLB
  • Serum samples of 94, 63, and 44 patients with AD, FTD, and DLB were collected, and serum samples of 197 normal healthy subjects were collected.
  • the antigen combinations in Table 5 were used to perform sensitivity and specificity tests on the above samples.
  • Example 2 the definition method of the positive and negative of a single antigen is referred to Example 2 and Example 3.
  • the overall sensitivity and specificity of the antigen combination are defined as follows:
  • the serum sample when a serum sample uses any one of the antigens in the combination to obtain a positive detection signal, the serum sample is a positive sample; otherwise, the serum sample is a negative sample. Based on the above positive and negative definition methods, the positive and negative results obtained by the antigen combination test are obtained, and then the sensitivity of the antigen combination in the patient sample is calculated.
  • sensitivity refers to the proportion of AD (or FTD, or DLB) patient samples that are correctly judged as positive, that is, the number of samples judged as positive in all AD (or FTD, or DLB) patient samples divided by the total number of all AD (or FTD, or DLB) patient samples; specificity refers to the proportion of healthy subject samples that are correctly judged as negative, that is, the number of healthy subject samples judged as negative in healthy subject samples divided by the total number of healthy subject samples.
  • Example 5 Demonstration of the effect of candidate antigen combination and corresponding full-length protein combination in detecting AD, FTD and DLB
  • Example 2 Referring to the detection method of Example 2, each sample in Example 4 was tested for AD, FTD, DLB sensitivity and AD specificity using each group of antigen combinations in Table 7. The results are shown in Table 8.

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Abstract

提供一种含有SERF2的蛋白抗原组合及其在制备用于区分阿尔兹海默症与额颞叶痴呆和路易体痴呆的产品中的用途。

Description

含有SERF2的蛋白抗原组合及其应用 技术领域
本发明属于生物检测领域，具体涉及一种用于区分阿尔茨海默症与额颞叶痴呆和路易体痴呆，检测自身抗体的含有SERF2蛋白抗原组合及其应用。
背景技术
阿尔茨海默症(Alzheimer＇s disease，简称AD)是以记忆和认知功能障碍为主要特征的进行性神经变性疾病，多发于老年群体，病程缓慢且不可逆。AD是最常见的痴呆类型，在所有痴呆患者中占比60～70％。目前AD的诊断方法主要为通过MMSE评分、MRI、CT和PET等影像学检测结合临床症状进行判断。
自身抗体是由个体免疫系统产生的、针对个体自身蛋白抗原的抗体。正常情况下，免疫系统响应体内的外源性蛋白或物质而产生抗体，但有时也会识别机体的一种或多种内源性组分，导致产生自身抗体。已有大量证据证实，血清中存在多种自身抗体参与神经性疾病和综合症。我们在中国专利CN 110850104 B公布了一系列可以用于AD患者自身抗体检测的抗原蛋白(和/或片段)及其组合。这些组合临床上用于诊断阿尔茨海默症(AD)可以取得很好的效果，但尚未涉及到AD与路易体痴呆(dementia with Lewy body,DLB)和额颞叶痴呆(Frontal Dementia,FTD)等相关痴呆的区分鉴别诊断。
路易体痴呆(dementia with Lewy body,DLB)是最常见的神经变性疾病之一；其主要病理特征为路易氏体(Lewy body，LB)广泛分布于患者的大脑皮层及脑干。据数据统计，65岁以上老年人的DLB患病率为3.6～6.6％，占痴呆患者10～20％。DLB临床症状和病理表现与AD与许多相似性，且40％以上的AD患者脑内会发现LB，证明AD和DLB有较高的重叠。另一方面，在临床表现上，DLB和AD均出现进行性认知功能 障碍，且在严重程度上难以区分；检测脑脊中的Tau蛋白和β淀粉样蛋白含量，两者也都有相似的变化。因此，这两种疾病在临床上的区分诊断极为困难。目前，临床上主要通过影像学检查来进行区分鉴别。一是通过颅脑MRI检查，两者脑萎缩的区域有细微的区别；二是通过脑血流SPECT/PET检查，通过影像学变化来区分AD和DLB。现有区分方法都对医生的经验要求较高。
额颞叶痴呆(FTD)是一种隐匿起病、进行性的神经变性疾病，是以额颞叶萎缩为特征的痴呆综合征，约占所有痴呆类型的5％～15％。临床上，FTD和AD患者均出现不同程度的认知功能障碍、行为障碍和语言障碍，并且FTD患者的MMSE评分与AD患者没有明显差异。目前一般通过CT、MRI进行FTD和AD的鉴别，AD可见广泛性脑萎缩，额颞痴呆显示额和(或)颞叶萎缩；但是仍需进一步组织病理学检查。
现有研究中，利用生物标志物区分上述疾病尚不可能。Paterson等人(Alzheimer's Research&Therapy(2018)10:32)检测了多种痴呆患者脑脊液中Tau蛋白，β淀粉样蛋白(Aβ)不同片断及其前体蛋白(APP)、NFL和YKL-40等10种蛋白的含量，以及相关蛋白含量的比例，结果证明这些标志物均不能有效鉴别区分AD和DLB；其中Aβ42/40对鉴别区分AD和行为变异型额颞叶痴呆(bvFTD)有一定的参考意义。但其不足之处是使用脑脊液样本对病人有一定风险，通常为病人所排斥。
同时，对比尸检结果，即使是在专业的痴呆诊疗中心，临床上AD的诊断仍然有25～30％的误诊率。AD是一种进行性的神经退行性疾病，症状随着病程增加逐渐加重，病程长，并且在发病早期进展缓慢。因此，越早进行诊断和干预，疾病越容易得到控制，病人也越容易受益。但是，越是在疾病早期，由于病理上的变化尚不明显，MRI和PET等仪器检测的准确率越低。并且，在疾病早期，AD与FTD，DLB等痴呆的临床症状很相似，也进一步加大了临床诊断的难度。
因此，开发一种能够有效区分鉴别AD与FTD、DLB等痴呆的诊断手段，具有重要的现实意义。
发明内容
本发明的目的是提供一种能够有效区分鉴别AD与FTD、DLB等痴呆，检测自身抗体的蛋白抗原组合及其应用。
为实现上述发明目的，本发明所采用的技术方案是：一种抗原组合，所述抗原组合至少包括SERF2蛋白和/或SNAP25蛋白。
优选的，所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示，所述SNAP25蛋白的氨基酸序列如SEQ ID NO：8所示。
优选的，同时包括SERF2蛋白、SNAP25蛋白。
优选的，还包括MAPT蛋白片段、RAGE蛋白片段、ASXL1蛋白片段、JMJD2D蛋白片段、P21蛋白片段、DNAJC8蛋白片段中的任意一种或多种。
优选的，所述MAPT蛋白片段的氨基酸序列如SEQ ID NO：1所示，所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
相应的，所述抗原组合在制备检测/鉴定阿尔茨海默症产品中的应用。
相应的，所述抗原组合在制备区分阿尔茨海默症、额颞叶痴呆、路易体痴呆的产品中的应用。
相应的，利用所述抗原组合制备的检测阿尔茨海默症的试剂盒。
相应的，SERF2蛋白和/或SNAP25蛋白和/或MAPT蛋白片段和/或RAGE蛋白片段和/或ASXL1蛋白片段和/或JMJD2D蛋白片段和/或P21蛋白片段和/或DNAJC8蛋白片段在制备检测/鉴定阿尔茨海默症产品中的应用，所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示；所述SNAP25 蛋白的氨基酸序列如SEQ ID NO：8所示；所述MAPT蛋白片段的氨基酸序列如SEQ ID NO：1所示；所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
相应的，SERF2蛋白和/或SNAP25蛋白和/或MAPT蛋白片段和/或RAGE蛋白片段和/或ASXL1蛋白片段和/或JMJD2D蛋白片段和/或P21蛋白片段和/或DNAJC8蛋白片段在制备区分阿尔茨海默症、额颞叶痴呆、路易体痴呆的产品中的应用，所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示；所述SNAP25蛋白的氨基酸序列如SEQ ID NO：8所示；所述MAPT蛋白片段的氨基酸序列如SEQ ID NO：1所示；所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
本发明具有以下有益效果：本发明提供了一种全新的蛋白抗原组合物，不仅可以有效鉴定AD患者，而且可以有效区分AD、FTD与DLB，实现准确AD的准确鉴定，具有重要的应用价值和研究价值。
具体实施方式
下面结合具体实施例对本申请进行进一步阐释。如无特殊说明，下述实施例中所使用的实验方法均为常规方法；所用的材料、试剂等均可从商业途径得到；所获得的数据均为进行至少3次重复后获得的平均值，且各重复获得的均为有效数据。
实施例一：抗原的重组载体构建、表达和纯化
1、抗原的选择。选择23种与阿尔茨海默症高度相关的抗原蛋白进行构建、表达和纯化。其分别的数据库ID具体如表1所示。
表1待测蛋白的数据库ID对应表
2、抗原重组载体的构建和表达。以人类cDNA文库(购自Invitrogen公司)或者全基因合成DNA为模板，分别设计引物，通过PCR、酶切、连接等分子克隆手段，将所述蛋白的全长基因克隆到pET28质粒上。同时，在蛋白N-端增加HIS、c-myc等标签，形成融合蛋白。将得到的重组表达载体通过DNA测序鉴定，确认包含正确的蛋白基因片段。需要说明的是，添加的标签只是方便识别和提取蛋白，并未对蛋白作为抗原时的功能产生决定性影响，使用时，不添加标签或根据需要添加其它标签均可。
将上述包含表1的蛋白基因片段的重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中，挑取克隆接种至LB培养基中，37℃摇床培养。当菌体密度达到OD600约为0.8时，降温至16℃，在每个LB培养基中加入0.1mM异丙基硫代-β-D-半乳糖苷(IPTG)，诱导表达过夜，获得菌体。
3、抗原的纯化。离心收集诱导表达的所述菌体，用PBS漂洗两遍。用裂解液(每g菌体加5～10mL裂解液)重悬并分散菌体，冰浴，超声破碎菌体(超声功率200W，破碎5S，休息5S)。破碎后13000rpm，10℃，离心20分钟，取上清，经Ni柱亲和层析和分子筛层析两步纯化后，采用SDS-PAGE电泳分析，确认蛋白的分子量、纯度，用Bradford法测定 浓度后，保存在-80℃备用。即获得纯化后的待测蛋白。
实施例二：从待测蛋白中筛选获得候选抗原
1、本实施例中使用的溶液和试剂如下：
(1)包被缓冲液为PBS缓冲液，pH＝7.4。其制备方法为：准确称取3.58g Na2HPO4·12H2O、0.23g KH2PO4·2H2O、0.2g KCl、8.0g NaCl，溶解于水中，清水定容至1L。
(2)封闭液/样本稀释液/抗体稀释液：将10g BSA(牛血清白蛋白)溶解于包被缓冲液中，清水定容至1L。
(3)洗涤液：现配现用。使用前在包被缓冲液中加入0.5％Tween20(V/V)，pH＝7.4。
(4)TMB显色剂，购自KPL公司。
(5)终止液：1M盐酸。
2、固相包被待测蛋白。用包被缓冲液将实施例一获得的纯化后的待测蛋白稀释至5μg/ml，加至96孔板，每孔50μl，4℃包被过夜。次日倒掉溶液，甩干，用洗涤液洗三次，每次每孔200μl。然后每孔加入200μl封闭液，室温孵育1h后，将封闭液倒掉，甩干，再用洗涤液洗三次，每次每孔200μl，并再次甩干；获得位于96孔板中的固相包被的抗原。
3、加入待测样本。将待测人血清用样本稀释液稀释100倍后，加入所述含待测蛋白的96孔板，每孔加稀释后的待测样本50μl。随后将96孔板置于微孔板振荡仪上，室温振荡孵育1h；甩干，用洗涤液洗三次，每次每孔200μl，并再次甩干。
4、加入酶标二抗。将1.0mg/ml辣根过氧化物酶标记的重组羊抗人免疫球蛋白G抗体(购自Jackson ImmunoResearch Inc.)用抗体稀释液稀释20000倍后，加入步骤3处理后的96孔板中，每孔加50μl。随后将96孔板置于微孔板振荡仪上，室温振荡孵育0.5h，将板甩干，用洗涤液洗三次，每次每孔200μl，并再次甩干。
5、显色反应及光密度值读数。在步骤4处理后的96孔板中，每孔加50μl TMB显色剂，振荡15s，室温下避光反应15min，再加入50μl终止液；然后用酶标仪读取450nm波长的吸收值，获得每个待测样本的检测信号(S)。
6、敏感性和特异性分析。分别取180例阳性样本(确诊为阿尔兹海默症患者的血清)和180例阴性样本(健康受试者血清)，按上述方法(450nm波长的吸收值)测定每个样本的检测信号(S)。以阴性样本为阴性参考样本，计算所有阴性参考样本检测信号(S)的平均值(M)及标准差(SD)，以M+3SD为Cut Off值。将检测信号(S)≥Cut Off值的样本(S≥M+3SD)定为阳性；将检测信号(S)＜Cut Off值的样本(S＜M+3SD)定为阴性。
基于样本阳性和阴性结果计算特异性和敏感性。其中，特异性是指健康受试者样本被正确地判定为阴性的比例，即在阴性样本中被正确地判定为阴性的数量除以阴性样本总数。敏感性是指阿尔兹海默症患者样本被正确地判定为阳性的比例，即在阳性样本中被判定为阳性的数量除以阳性样本总数。通过计算得出使用每个被测蛋白作为抗原进行样本检测时的敏感性和特异性，结果如表2所示。
表2各被测蛋白/蛋白片段作为抗原的敏感性和特异性展示表

7、从上述各被测蛋白或片段中选择筛选出敏感性≥15％且特异性≥85％的蛋白作为候选抗原。筛选结果如表3所示。
表3从被测蛋白中筛选出的候选抗原展示表
实施例三：候选抗原检测DLB和FTD病人样本效果展示
分别取43例DLB阳性样本(确诊为DLB患者的血清)、63例FTD阳性样本(确诊为FTD患者的血清)和232例阴性样本(健康受试者血清)，按照实施例二的方法，以表3中的候选抗原(蛋白质片段)来测定每个样本的检测信号(S)。以阴性样本为阴性参考样本，计算所有阴性参考样本检测信号(S)的平均值(M)及标准差(SD)，以M+3SD为Cut Off值。将检测信号(S)≥Cut Off值的样本(S≥M+3SD)定为阳性；将检测信号(S)＜Cut Off值的样本(S＜M+3SD)定为阴性。
基于样本阳性和阴性结果计算特异性和敏感性。其中，特异性是指健康受试者样本被正确地判定为阴性的比例，即在阴性样本中被正确地判定为阴性的数量除以阴性样本总数。DLB样本敏感性是指DLB患者样本被正确地判定为阳性的比例，即在DLB阳性样本中被判定为阳性的数量除以阳性样本总数。FTD样本敏感性是指FTD患者样本被正确地判定 为阳性的比例，即在FTD阳性样本中被判定为阳性的数量除以阳性样本总数。通过计算得出使用每个被测蛋白作为抗原进行样本检测时的敏感性和特异性，结果如表4所示。
表4 DLB和FTD病人样本测试结果展示
实施例四：候选抗原组合检测AD、FTD和DLB的效果展示
1、从表3的各候选蛋白抗原中选择蛋白质或蛋白质片段，形成包含SERF2和SNAP25的不同抗原组合，具体组合方式如表5所示。
表5各抗原组合对照表


2、分别取AD、FTD、DLB患者血清样本94、63、44例，取正常的健康受试者血清样本197例。参照实施例二的检测方法，分别使用表5中各组抗原组合对上述各样本进行敏感性和特异性检测。
抗原组合中，单个抗原的阳性和阴性的定义方法参照实施例二和实施例三。抗原组合的整体敏感性和特异性定义方法如下：
对于一个抗原组合，当某个血清样本采用该组合中的任意一个抗原得到的检测信号为阳性检测信号时，该血清样本为阳性样本；否则该血清样本为阴性样本。基于上述阳性和阴性定义方法，检测获得一个抗原组合检测得到的阳性和阴性结果，进而计算该抗原组合在患者样本中的敏感性。
其中，敏感性是指在AD(或FTD、或DLB)患者样本总共被正确地判定为阳性的比例，即在所有AD(或FTD、或DLB)患者样本中被判定为阳性的数量除以所有AD(或FTD、或DLB)患者样本总数；特异性是指健康受试者样本被正确地判定为阴性的比例，即在健康受试者样本中，健康受试者样本中被判定为阴性的数量除以健康受试者样本总数。
各组抗原组合检测获得的敏感性和特异性结果如表6所示。
表6各组抗原组合检测结果对照表


从表6的结果可以看出，各个组合检测的特异性都很好，均在81％以上，部分组合达到85％乃至90％以上；对阿尔茨海默症(AD)患者的检出敏感性相对于额颞痴呆(FTD)和路易体痴呆(DLB)的检出敏感性要高出数倍。比如，组合13对AD的敏感性达到72.34％，而对FTD检出敏感性只有7.94％，对DLB的检出敏感性也非常低，仅有4.55％，具有很好的鉴别区分意义。在后期应用时，还可根据需要，从本发明提供的候选抗原或抗原组合中选择一种或多种，制作成试剂盒，用于AD的诊断，和/或区分鉴别FTD与DLB。
实施例五：候选抗原组合与对应的全长蛋白组合检测AD、FTD和DLB的效果展示
1、从实施例四的表5中选择4组不同的抗原组合，蛋白质或蛋白质片段均来自于表3；并基于组1～4的抗原组合中各蛋白质片段对应的全长蛋白，设置对应的全长蛋白组合，分别为对照组1～4，具体如表7所示。如果表3中的蛋白质已经是全长蛋白，则该蛋白质不做调整。
表7各抗原组合对照表

2、参照实施例二的检测方法，分别使用表7中各组抗原组合对实施例四中各样本进行AD、FTD、DLB敏感性和AD特异性检测。结果如表8所示。
表8各组抗原组合检测结果对照表
从表8的结果可以看出，相对于完全使用全长蛋白的组合，包含片断的各个组合检测的特异性都较好，均在81％以上；同时，包含片断的各个组合不仅保持了对AD的高检出敏感性，其对FTD，DLB的检出敏感性也更低，具有更好的鉴别区分意义。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形、变型、修改、替换，均应落入本发明权利要求书确定的保护范围内。

Claims (10)

  1. 一种抗原组合，其特征在于：所述抗原组合至少包括SERF2蛋白和/或SNAP25蛋白。
  2. 根据权利要求1所述的抗原组合，其特征在于：所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示，所述SNAP25蛋白的氨基酸序列如SEQ ID NO：8所示。
  3. 根据权利要求1或2所述的抗原组合，其特征在于：同时包括SERF2蛋白、SNAP25蛋白。
  4. 根据权利要求1～3任意一项所述的抗原组合，其特征在于：还包括MAPT蛋白片段、RAGE蛋白片段、ASXL1蛋白片段、JMJD2D蛋白片段、P21蛋白片段、DNAJC8蛋白片段中的任意一种或多种。
  5. 根据权利要求4所述的抗原组合，其特征在于：所述MAPT蛋白片段的氨基酸序列如SEQ ID NO：1所示，所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
  6. 权利要求1～5任意一项所述抗原组合在制备检测/鉴定阿尔茨海默症产品中的应用。
  7. 权利要求1～5任意一项所述抗原组合在制备区分阿尔茨海默症、额颞叶痴呆、路易体痴呆的产品中的应用。
  8. 利用权利要求1～5任意一项所述抗原组合制备的检测阿尔茨海默症的试剂盒。
  9. SERF2蛋白和/或SNAP25蛋白和/或MAPT蛋白片段和/或RAGE蛋白片段和/或ASXL1蛋白片段和/或JMJD2D蛋白片段和/或P21蛋白片段和/或DNAJC8蛋白片段在制备检测/鉴定阿尔茨海默症产品中的应用，其特征在于：所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示；所述SNAP25蛋白的氨基酸序列如SEQ ID NO：8所示；所述MAPT蛋白片 段的氨基酸序列如SEQ ID NO：1所示；所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
  10. SERF2蛋白和/或SNAP25蛋白和/或MAPT蛋白片段和/或RAGE蛋白片段和/或ASXL1蛋白片段和/或JMJD2D蛋白片段和/或P21蛋白片段和/或DNAJC8蛋白片段在制备区分阿尔茨海默症、额颞叶痴呆、路易体痴呆的产品中的应用，其特征在于：所述SERF2蛋白的氨基酸序列如SEQ ID NO：7所示；所述SNAP25蛋白的氨基酸序列如SEQ ID NO：8所示；所述MAPT蛋白片段的氨基酸序列如SEQ ID NO：1所示；所述RAGE蛋白片段的氨基酸序列如SEQ ID NO：4所示，所述ASXL1蛋白片段的氨基酸序列如SEQ ID NO：5所示，所述JMJD2D蛋白片段的氨基酸序列如SEQ ID NO：6所示，所述P21蛋白片段的氨基酸序列如SEQ ID NO：2所示，所述DNAJC8蛋白片段的氨基酸序列如SEQ ID NO：3所示。
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