TW201534614A - Peptides for detecting anti-[alpha] 7 nAChR antibody - Google Patents

Peptides for detecting anti-[alpha] 7 nAChR antibody Download PDF

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TW201534614A
TW201534614A TW103107997A TW103107997A TW201534614A TW 201534614 A TW201534614 A TW 201534614A TW 103107997 A TW103107997 A TW 103107997A TW 103107997 A TW103107997 A TW 103107997A TW 201534614 A TW201534614 A TW 201534614A
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antibody
binding molecule
sequence
alpha
antibody binding
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TWI606060B (en
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Kuang-Hui Lee
Jun Wei
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Glory Biomedical Co Ltd
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Abstract

The present invention provides neuronal acetylcholine receptor subunit alpha-7 polypeptides designed to specifically bind to neuronal acetylcholine receptor subunit alpha-7 antibody. Said neuronal acetylcholine receptor subunit alpha-7 polypeptide could help detect anti-neuronal acetylcholine receptor subunit alpha-7 antibody in patients, and thus diagnose schizophrenia early.

Description

用於檢測神經性乙醯膽鹼受器阿爾法七次單位抗體之多肽分子 Polypeptide molecule for detecting alpha seven-unit antibody of neurothyroid acetylcholine receptor

本發明關於一種用於檢測抗體之多肽分子,特別是關於一種用於檢測神經性乙醯膽鹼受器阿爾法7次單位(neuronal acetylcholine receptor subunit alpha-7,簡稱α 7 nAChR)抗體之多肽分子。 The present invention relates to a polypeptide molecule for detecting an antibody, and more particularly to a polypeptide molecule for detecting a neuronal acetylcholine receptor subunit alpha-7 ( α 7 nAChR) antibody.

隨著社會變遷,生活步調日趨緊湊,壓力伴隨而生,使得各種精神疾病(Psychiatric disorder)之發病率都有上升之趨勢。其中,精神分裂症(Schizophrenia)是一種較為常見且嚴重的精神疾病,其終身患病率為1%。 With the changes of society, the pace of life is becoming more and more compact, and the pressure is accompanied, which makes the incidence of various Psychiatric disorders increase. Among them, Schizophrenia is a relatively common and serious mental illness with a lifetime prevalence of 1%.

多年來,醫生以臨床觀察及會談的方式獲取病史及精神狀態等訊息,進而依據症狀學判斷個體是否罹患精神分裂症。然而,這種診斷方式受主觀因素影響甚鉅,不可避免地常發生誤診和漏診的情形,為目前精神分裂症疾病診斷方法飽受批評的重要原因之一。因此,亟需一種精神分裂症之客觀檢測指標,作為臨床醫生綜合評估是否罹患精神分裂症之輔助依據。 Over the years, doctors have obtained information such as medical history and mental state through clinical observation and interviews, and then judged whether individuals suffer from schizophrenia based on symptomology. However, this type of diagnosis is greatly influenced by subjective factors. Inevitably, misdiagnosis and missed diagnosis often occur, which is one of the important reasons for the current criticism of schizophrenia disease diagnosis methods. Therefore, an objective detection index for schizophrenia is urgently needed as a supplementary basis for clinicians to comprehensively evaluate whether or not they suffer from schizophrenia.

有鑑於習知技術的缺陷,本發明提供一種製程簡單之神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子,可與神經性乙醯膽鹼受器阿爾法7次單位抗體結合,以檢測個體血液、血漿、或血清中的神經性乙醯膽鹼受器阿爾法7次單位抗體(即自身抗體)含量,進而早期診斷是否罹患精神分裂症。 In view of the defects of the prior art, the present invention provides a linear polypeptide molecule of a simple 7-unit unit epitope of a neurogenic acetylcholine receptor, which can be combined with a neurogenic acetylcholine receptor alpha 7-unit antibody. To detect the content of alpha 7-unit antibody (ie, autoantibody) in the blood, plasma, or serum of an individual, and to diagnose early schizophrenia.

另外,本發明提供一種製備成本較低且易於保存之神經性乙醯膽鹼受器阿爾法7次單位多肽分子,使所製備出的檢測試劑及免疫酵素測定套組易於保存。 In addition, the present invention provides a neurogenic acetylcholine receptor alpha 7-unit polypeptide molecule which is low in cost and easy to preserve, and the prepared detection reagent and immunoenzyme assay kit are easy to store.

本發明亦提供一種便於檢測精神分裂症之試劑及免疫酵素測定套組,以增加使用之便利性及快速性。 The invention also provides a reagent and an immunoenzyme assay kit for facilitating detection of schizophrenia, thereby increasing the convenience and rapidity of use.

於一較佳實施例中,本發明提供一種抗體結合分子,可與一神經性乙醯膽鹼受器阿爾法7次單位(Neuronal acetylcholine receptor subunit alpha-7)之一抗體結合;其中,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 In a preferred embodiment, the present invention provides an antibody binding molecule which binds to an antibody of a neuronal acetylcholine receptor subunit alpha-7; wherein the antibody binds The molecule is 25-40 amino acids in length, and the sequence of the antibody binding molecule comprises at least one of SEQ ID NOS: 1 to 5.

於一較佳實施例中,該抗體結合分子之序列長度為25至35個胺基酸。 In a preferred embodiment, the antibody binding molecule has a sequence length of from 25 to 35 amino acids.

於一較佳實施例中,該抗體結合分子序列中的脯胺酸(Proline)數量為0至3個。 In a preferred embodiment, the amount of proline in the antibody binding molecule sequence is from 0 to 3.

於一較佳實施例中,該抗體結合分子序列中的麩胺酸(Glutamic acid)數量為0至3個,抑或是,其序列中的半胱胺酸(Cysteine)數量 為0至1個。 In a preferred embodiment, the amount of glutamic acid (Glutamic acid) in the antibody binding molecule sequence is 0 to 3, or the number of Cysteine in the sequence. It is 0 to 1.

於一較佳實施例中,該抗體結合分子係線性多肽分子,抑或是,該抗體結合分子於一水溶液或一血漿中不形成阿爾法螺旋(alpha helix)結構。 In a preferred embodiment, the antibody binds to a linear polypeptide molecule of the molecule, or the antibody binding molecule does not form an alpha helix structure in an aqueous solution or a plasma.

於一較佳實施例中,該抗體結合分子與該抗體間的結合力,大於該抗體與一洗滌緩衝液間的作用力。 In a preferred embodiment, the binding strength of the antibody binding molecule to the antibody is greater than the interaction between the antibody and a wash buffer.

本發明更提供一種檢測試劑,包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 The present invention further provides a detection reagent comprising an antibody binding molecule having a length of 25-40 amino acids, and the sequence of the antibody binding molecule comprises at least one of SEQ ID NOS: 1 to 5.

本發明再提供一種免疫酵素測定套組(ELISA Kit),包含一試劑;其中,該試劑中更包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 The invention further provides an immunoassay assay kit (ELISA Kit) comprising a reagent; wherein the reagent further comprises an antibody binding molecule, the antibody binding molecule has a length of 25-40 amino acids, and the antibody binds The sequence of the molecule comprises at least one of SEQ ID NOS: 1 to 5.

於一較佳實施例中,該試劑中之該抗體結合分子濃度為0.75μg/mL~5mg/mL。 In a preferred embodiment, the concentration of the antibody binding molecule in the reagent is from 0.75 μg/mL to 5 mg/mL.

本發明又提供一種免疫酵素測定套組(ELISA Kit),包含一多孔盤;其中,該多孔盤中包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 The invention further provides an immunological enzyme assay kit (ELISA Kit) comprising a porous disk; wherein the porous disk comprises an antibody binding molecule having a length of 25-40 amino acids, and the antibody binding molecule The sequence of the antibody binding molecule comprises at least one of SEQ ID NOS: 1 to 5.

於一較佳實施例中,該免疫酵素測定套組係用於檢測一血液,一血漿,或一血清中的一神經性乙醯膽鹼受器阿爾法7次單位之抗體含量。 In a preferred embodiment, the immunoenzyme assay kit is used to detect the antibody content of an alpha 7-unit unit of a neurotransmitter in a blood, a plasma, or a serum.

於一較佳實施例中,該免疫酵素測定套組可用於早期診斷是否罹患一精神疾病或作為追蹤一精神疾病病況之指標。 In a preferred embodiment, the immunoenzyme assay kit can be used for early diagnosis of a mental illness or as an indicator of a condition of a mental illness.

於一較佳實施例中,該精神疾病係精神分裂症。 In a preferred embodiment, the mental illness is schizophrenia.

圖一:依精神分裂症群組及其匹配健康群組之SBI值所畫出的接收者操作特徵曲線。 Figure 1: Receiver operating characteristic curves plotted against the SBI values of the schizophrenia group and its matching healthy group.

精神分裂症係一種神經精神缺失疾病(Neuropsychiatric disorder)。雖然,人們對於其致病原因仍不甚清楚,但已知精神分裂症與個體免疫功能紊亂、自體免疫反應及發炎反應有密切關係(Strous and Shoenfeld,2006;Potvin et al.,2008),且精神分裂症病人體內的細胞免疫功能下降,體液免疫功能卻顯著增加(Potvin et al.,2008)。 Schizophrenia is a neuropsychiatric disorder. Although the cause of the disease is still unclear, it is known that schizophrenia is closely related to individual immune dysfunction, autoimmune response and inflammatory response (Strous and Shoenfeld, 2006; Potvin et al., 2008). Moreover, the cellular immune function of patients with schizophrenia is decreased, and the humoral immune function is significantly increased (Potvin et al., 2008).

阿爾法7尼古丁受器(Alpha-7 nicotinic receptor,或稱α7 receptor)係一種離子通道型受器,對鈣離子有很高的選擇性,由五個神經性乙醯膽鹼受器阿爾法7次單位(Neuronal acetylcholine receptor subunit alpha-7,簡稱α 7 nAChR,係CHRNA7基因表現後的蛋白質終產物)組成,表現於大腦皮質、大腦海馬迴(Hippocampus;Hellstrom-Lindahl et al.,1999)、及淋巴球細胞(Lymphocyte;Wang et al.,2001;Villiger et al.,2002),在神經元間的訊號傳遞、學習、記憶、突觸的可塑性、及免疫反應上都扮演了重要角色。 Alpha-7 nicotinic receptor (α7 receptor) is an ion channel type receptor with high selectivity for calcium ions. (Neuronal acetylcholine receptor subunit alpha-7, abbreviated as α 7 nAChR, which is a protein end product expressed after CHRNA7 gene expression), expressed in the cerebral cortex, hippocampus (Hippocampus; Hellstrom-Lindahl et al., 1999), and lymphocytes Cells (Lymphocyte; Wang et al., 2001; Villiger et al., 2002) play an important role in signal transmission, learning, memory, synaptic plasticity, and immune response between neurons.

有研究顯示,神經性乙醯膽鹼受器阿爾法7次單位基因(簡稱CHRNA7 gene)缺失與精神分裂症有密切的關係(Shinawi et al.,2009)。此外,更有23%的精神分裂症患者體內的神經性乙醯膽鹼受器阿爾法7次單位(α 7 nAChR)自身抗體(Autoantibody)明顯高於一般人(Chandley et al.,2009),可能阻斷尼古丁受體功能,進而參與在精神分裂症發病的過程中。據此,依發明人多年的研究成果及經驗,認為應能建立一種簡便、精確之檢驗方法,透過檢測血液中之α 7 nAChR自身抗體含量,早期診斷精神分裂症疾病。 Studies have shown that the loss of the neuroic acetylcholine receptor alpha 7-unit gene (CHRNA7 gene) is closely related to schizophrenia (Shinawi et al., 2009). In addition, 23% more schizophrenic patient neuropathic acetylcholine alpha 7 receptors subunits 7 nAChR) autoantibodies (autoantibody) was significantly higher than the average person (Chandley et al., 2009) , may be hindered Nicotine receptor function is broken, which in turn is involved in the pathogenesis of schizophrenia. Based on the research results and experience of the inventors for many years, it is believed that a simple and accurate test method should be established to detect schizophrenia disease early by detecting the content of α 7 nAChR autoantibodies in the blood.

以下係利用本發明之實施例之詳細說明書,以及本發明之技術、特點。然本實施例並非用以限定本發明,任何熟悉此技術者,在不脫離本發明之精神和範圍內所作之各種更動、潤飾,均應包含在本發明之申請專利範圍內。 The following is a detailed description of embodiments of the invention, as well as the techniques and features of the invention. The present invention is not intended to limit the invention, and any changes and modifications made by those skilled in the art without departing from the spirit and scope of the invention are included in the scope of the invention.

本發明提供至少一種抗體結合分子,可與一神經性乙醯膽鹼受器阿爾法7次單位(Neuronal acetylcholine receptor subunit alpha-7,簡稱α 7 nAChR)之抗體結合,以檢測個體血液、血漿、或血清中的神經性乙醯膽鹼受器阿爾法7次單位之抗體(即自身抗體)含量,進而診斷是否罹患精神分裂症或作為追蹤精神分裂症病況之指標。 The present invention provides at least one antibody binding molecule, which can be combined with an antibody of Neuronal acetylcholine receptor subunit alpha-7 ( α 7 nAChR) to detect blood, plasma, or The serum acetylcholine in the serum is subjected to an alpha 7-unit antibody (ie, autoantibody), which in turn diagnoses schizophrenia or serves as an indicator for tracking schizophrenia.

本案之抗體結合分子可以為神經性乙醯膽鹼受器阿爾法7次單位重組蛋白(α 7 nAChR Recombinant)、神經性乙醯膽鹼受器阿爾法7次單位表位(Epitope)多肽分子、或神經性乙醯膽鹼受器阿爾法7次單位表位(Epitope)線性多肽分子。其中,相較於神經性乙醯膽鹼受器阿爾法7次單位重組蛋白需要經過載體構建、轉染、表達、篩選、純化等 步驟,且重組蛋白空間結構較複雜,較佳者,為神經性乙醯膽鹼受器阿爾法次單位7表位線性多肽分子(以下部分內容將簡稱為α 7 nAChR表位線性多肽分子)。 The antibody binding molecule of the present invention may be a neurogenic acetylcholine receptor alpha 7 nARR Recombinant, a neurogenic acetylcholine receptor alpha 7 subunit epitope (Epitope) polypeptide molecule, or a nerve A acetylcholine receptor alpha alpha epitope linear polypeptide molecule. Among them, compared with the neurogenic acetylcholine receptor, the 7-unit recombinant protein needs to be constructed, transfected, expressed, screened, purified, etc., and the spatial structure of the recombinant protein is complex, preferably, neurotic. Acetylcholine receptor alpha subunit 7 epitope linear polypeptide molecule (hereinafter part will be referred to as α 7 nAChR epitope linear polypeptide molecule).

由於抗原抗體在空間結構和空間構型上互補,兩者之結合實際上只發生在抗原表位(Epitope)和抗體結合位(Paratope)之間。因此,僅抗原表位片段就可以代表整個蛋白與抗體結合的狀態及親和特性。依發明人多年之經驗及研究結果,認為應能從神經性乙醯膽鹼受器阿爾法7次單位的序列及結構中,找出神經性乙醯膽鹼受器阿爾法7次單位表位,並從眾多神經性乙醯膽鹼受器阿爾法7次單位表位序列中篩選出適當的「α 7 nAChR表位線性多肽分子」,以發展快速、有效、穩定的精神分裂症檢測試劑及精神分裂症免疫酵素測定套組(ELISA Kit)。 Since antigenic antibodies are complementary in spatial and spatial configuration, the combination of the two actually occurs only between the epitope (Epitope) and the antibody binding site (Paratope). Therefore, only the epitope fragment can represent the state and affinity of the entire protein to bind to the antibody. According to the inventor's many years of experience and research results, it is believed that the 7-unit epitope of the neurogenic acetylcholine receptor can be found from the sequence and structure of the 7-unit unit of the neuroacetylcholine receptor. Screening appropriate " α 7 nAChR epitope linear polypeptide molecules" from a variety of neurogenic acetylcholine receptor alpha 7-unit epitope sequences to develop rapid, effective, and stable schizophrenia detection reagents and schizophrenia Immunoassay kit (ELISA Kit).

神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子的設計:Neuroic acetylcholine receptor alpha 7-unit epitope linear polypeptide molecule design:

發明人利用生物資訊學分析神經性乙醯膽鹼受器阿爾法7次單位之胺基酸序列,再經多個表位預測軟體比較抗原性相關參數,進而挑選出候選序列。較佳者,該些候選序列之長度為25~40個胺基酸,抑或是,較佳者,該些候選序列之長度為25~35個胺基酸。 The inventors used bioinformatics to analyze the amino acid sequence of the 7-unit unit of the neuroacetylcholine receptor, and then predicted the antigen-related parameters by multiple epitope prediction software, and then selected the candidate sequences. Preferably, the candidate sequences are 25 to 40 amino acids in length, or, preferably, the candidate sequences are 25 to 35 amino acids in length.

免疫酵素測定法(ELISA)具高度靈敏性,因此對胺基酸序列分子的穩定性要求極高。發明人進一步遵循以下7個原則篩選候選序列: The immunoenzymatic assay (ELISA) is highly sensitive and therefore requires a high degree of stability to the amino acid sequence molecule. The inventors further followed the following seven principles to screen candidate sequences:

1)選擇不形成阿爾法螺旋(alpha helix)結構的序列。較佳者,於一水溶液或一血漿中不形成阿爾法螺旋結構。 1) Select a sequence that does not form an alpha helix structure. Preferably, the alpha helix structure is not formed in an aqueous solution or a plasma.

2)兩端的肽段比中間的排列合理。 2) The peptides at both ends are arranged better than the middle.

3)避免蛋白內部重複。 3) Avoid internal repeats of the protein.

4)避免同源性強的肽段。 4) Avoid peptides with strong homology.

5)序列中儘量避免半胱胺酸(Cysteine)和麩胺酸(Glutamic acid)。較佳者,序列中的麩胺酸(Glutamic acid)數量為0至3個,抑或是,較佳者,序列中的半胱胺酸(Cysteine)數量為0至1個。 5) Avoid cysteine (Cysteine) and glutamic acid (Glutamic acid) in the sequence. Preferably, the number of glutamic acid in the sequence is from 0 to 3, or, preferably, the number of Cysteine in the sequence is from 0 to 1.

6)避免過多的脯胺酸(Proline),但序列中具有1-2個脯胺酸利於肽鏈結構穩定,對產生特異性抗體有益。較佳者,序列中的脯胺酸(Proline)數量為0至3個。 6) Avoid excessive proline (Proline), but having 1-2 proline in the sequence facilitates the stability of the peptide chain structure and is beneficial for the production of specific antibodies. Preferably, the number of proline in the sequence is from 0 to 3.

7)較佳者,須含有第二型人類白血球抗原(Human leukocyte antigen class II,簡稱HLA class II)系統的限制性表位元,包括HLA-DR、HLA-DP、以及HLA-DQ的限制性表位。這些表位可被90%以上華人群體的第二型人類白血球抗原系統所識別。 7) Preferably, it must contain a restriction epitope of the Human leukocyte antigen class II (HLA class II) system, including HLA-DR, HLA-DP, and HLA-DQ. gauge. These epitopes can be recognized by the second type human leukocyte antigen system of more than 90% of the Chinese population.

發明人最後篩選出下列五種神經性乙醯膽鹼受器阿爾法7次單位表位線性多狀分子,依序為序列表中的SEQ ID NO:1~5: The inventors finally screened the following five neurogenic acetylcholine receptor alpha 7-element epitope linear polymorphisms, which are SEQ ID NOs: 1 to 5 in the sequence listing:

SEQ ID NO 1:NH3 +- LTVYFSLSLLQIMDVLTTNIWLQMSWTDHYLQK -COO- SEQ ID NO 1: NH 3 + - LTVYFSLSLLQIMDVLTTNIWLQMSWTDHYLQK -COO -

SEQ ID NO 2:NH3 +- VSLQGEFQRKLYKELVKNYNPLERPVANDSQPL -COO- SEQ ID NO 2: NH 3 + - VSLQGEFQRKLYKELVKNYNPLERPVANDSQPL -COO -

SEQ ID NO 3:NH3 +- EMPKWTRVILLNWCAWFLRMKRPGED -COO- SEQ ID NO 3: NH 3 + - EMPKWTRVILLNWCAWFLRMKRPGED -COO -

SEQ ID NO 4:NH3 +- RLMAFSVFTIICTIGILMSAPNFVEAVSK -COO- SEQ ID NO 4: NH 3 + - RLMAFSVFTIICTIGILMSAPNFVEAVSK -COO -

SEQ ID NO 5:NH3 +- KNQVLTTNIWLQMSWTDHYLQWNVSCR -COO- SEQ ID NO 5: NH 3 + - KNQVLTTNIWLQMSWTDHYLQWNVSCR -COO -

製備神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子:Preparation of a linear acetylcholine receptor alpha 7-unit epitope linear polypeptide molecule:

以化學法合成該些神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子。較佳者,純度須大於95%。較佳者,pH值大於7。可委託胜肽合成服務公司合成該些α 7 nAChR表位線性多肽分子,例如委託明欣生物科技有限公司(地址:台北市南港區園區街3號10樓之1;電話:02-26557128;網址:http://www.missionbio.com.tw/)製備。 The neuropeptides of the alpha acetylcholine receptor are linearly synthesized by a chemical method. Preferably, the purity must be greater than 95%. Preferably, the pH is greater than 7. The peptide synthesis synthesis service company can be commissioned to synthesize the linear peptide molecules of the α 7 nAChR epitope, for example, entrusted Mingxin Biotechnology Co., Ltd. (Address: 1st Floor, 10th Floor, No. 3, Park Street, Nangang District, Taipei; Tel: 02-26557128; Http://www.missionbio.com.tw/) Preparation.

將合成的神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子溶於67%的乙酸中(5mg α 7 nAChR表位線性多肽分子/1mL之67%乙酸),於-20℃之低溫冰箱中保存。 The synthetic neuroacetylcholine receptor alpha 7-element epitope linear polypeptide molecule was dissolved in 67% acetic acid (5 mg α 7 nAChR epitope linear polypeptide molecule / 1 mL of 67% acetic acid) at -20 ° C Store in a low temperature freezer.

神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子結合性分析:Neuropeptide acetylcholine receptor alpha 7-unit epitope linear peptide molecular binding analysis:

利用免疫酵素測定法(ELISA)進行本案五種神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子與神經性乙醯膽鹼受器阿爾法7次單位抗體間的結合性。 The binding of the linear 7-element epitope linear polypeptide molecule to the neuroacetylcholine receptor alpha 7-unit antibody was performed by immuno-enzyme assay (ELISA).

首先,以0.01M PBS(pH7.0~7.4)將各種包被液濃度調整成7.5~12.5μg/mL。0.01M PBS係1公升去離子水中包含0.2964g NaH2PO4‧2H2O、2.9g Na2HPO4‧12H2O、8g NaCl。較佳者,該0.01M PBS中包含0.1% NaN3First, various coating liquid concentrations were adjusted to 7.5 to 12.5 μg/mL in 0.01 M PBS (pH 7.0 to 7.4). 0.01 M PBS in 1 liter of deionized water contained 0.2964 g of NaH 2 PO 4 ‧2H 2 O, 2.9 g of Na 2 HPO 4 ‧12H 2 O, 8 g of NaCl. Preferably, the 0.01 M PBS contains 0.1% NaN 3 .

其中,實驗抗原包被液1係包含本案SEQ ID NO:1之α 7 nAChR表位線性多肽分子。實驗抗原包被液2係本案SEQ ID NO:2之α 7 nAChR表位線性多肽分子。實驗抗原包被液3係本案SEQ ID NO:3之α 7 nAChR表位線性多肽分子。實驗抗原包被液4係本案SEQ ID NO:4之α 7 nAChR表位線性多肽分子。實驗抗原包被液5係本案SEQ ID NO:5之α 7 nAChR表位線性多肽分子。正對照抗原包被液係人類神經性乙醯膽鹼受器阿爾法7次單位重組蛋白(Cat.No.H00001139-P01,Abnova)。負對照抗原包被液係發明人自行設計的線性多肽分子,與人類神經性乙醯膽鹼受器阿爾法7次單位無同源性,故不會與乙醯膽鹼受器阿爾法7次單位之抗體交叉結合。 Wherein, the experimental antigen coating solution 1 comprises the α 7 nAChR epitope linear polypeptide molecule of SEQ ID NO: 1 of the present invention. The experimental antigen coating solution 2 is the α 7 nAChR epitope linear polypeptide molecule of SEQ ID NO: 2 of the present invention. The experimental antigen coating solution 3 is the α 7 nAChR epitope linear polypeptide molecule of SEQ ID NO: 3 of the present invention. The experimental antigen coating solution 4 is the α 7 nAChR epitope linear polypeptide molecule of SEQ ID NO: 4 of the present invention. The experimental antigen coating solution 5 is the α 7 nAChR epitope linear polypeptide molecule of SEQ ID NO: 5 of the present invention. Positive control antigen coating human neurogenic acetylcholine receptor alpha 7-unit recombinant protein (Cat. No. H00001139-P01, Abnova). Negative control antigen coating solution The linear polypeptide molecule designed by the inventor has no homology with the human neurogenic acetylcholine receptor alpha 7-unit, so it will not be associated with the acetylcholine receptor alpha 7 unit. Antibody cross-binding.

將上述各種包被液分別包被(Coating)於多孔盤,每孔100μL,於4℃環境靜置隔夜(Overnight)。較佳者,該多孔盤為96孔盤,但不以此為限。以96孔盤(Cat.No.224-0096,COSTAR)為例,於A列的2個孔中包被實驗抗原包被液1,是為實驗抗原組1。於B列的2個孔中包被實驗抗原包被液2,是為實驗抗原組2。於C列的2個孔中包被實驗抗原包被液3,是為實驗抗原組3。於D列的2個孔中包被實驗抗原包被液4,是為實驗抗原組4。於E列的2個孔中包被實驗抗原包被液5,是為實驗抗原組5。於F列的2個孔中包被正對照抗原包被液,是為正對照抗原組。於G列的2個孔中包被負對照抗原包被液,是為負對照抗原組。於H列的2個孔中包被0.01M PBS(pH7.0~7.4),是為負對照組。 Each of the above-mentioned various coating liquids was coated on a porous disk, 100 μL per well, and allowed to stand overnight at 4 ° C (Overnight). Preferably, the porous disk is a 96-well disk, but is not limited thereto. Taking the 96-well plate (Cat. No. 224-0096, COSTAR) as an example, the experimental antigen coating solution 1 was coated in two wells of column A, which was the experimental antigen group 1. The experimental antigen coating solution 2 was coated in two wells of column B, which was the experimental antigen group 2. The experimental antigen coating solution 3 was coated in two wells of the C column, which was the experimental antigen group 3. The experimental antigen coating solution 4 was coated in two wells of column D, which was the experimental antigen group 4. The experimental antigen coating solution 5 was coated in two wells of the E column, which was the experimental antigen group 5. A positive control antigen coating solution was coated in two wells of column F, which was a positive control antigen group. A negative control antigen coating solution was coated in two wells of column G, which was a negative control antigen group. 0.01 M PBS (pH 7.0 to 7.4) was coated in two wells of column H, which was a negative control group.

以0.01M PBS(另外添加0.05% TWEEN-20)清洗3次。亦即,以洗滌緩衝液清洗3次。 Wash 3 times with 0.01 M PBS (additionally 0.05% TWEEN-20). That is, it was washed 3 times with a washing buffer.

利用樣品稀釋分析液(0.01M PBS中添加1% BSA)將人類神經性乙醯膽鹼受器阿爾法7次單位多株抗體(LifeSpan BioSciences)稀釋 100~500倍,每孔加入100μL,25℃培養2~3小時。 Dilute human neurotransmitter acetylcholine receptor alpha 7-unit antibody (LifeSpan BioSciences) using sample dilution assay (1% BSA in 0.01 M PBS) 100 to 500 times, 100 μL per well, and culture at 25 ° C for 2 to 3 hours.

以0.01M PBS(另外添加0.005% TWEEN-20)清洗3遍。亦即,以洗滌緩衝液清洗3次。其中,CHRNA7表位線性多肽分子與神經性乙醯膽鹼受器阿爾法7次單位多株抗體間的結合力,大於神經性乙醯膽鹼受器阿爾法7次單位多株抗體與該洗滌緩衝液間的作用力,因此神經性乙醯膽鹼受器阿爾法7次單位多株抗體不會被洗滌緩衝液沖洗掉。 Wash 3 times with 0.01 M PBS (additionally 0.005% TWEEN-20). That is, it was washed 3 times with a washing buffer. Wherein, the binding ability of the linear polypeptide molecule of the CHRNA7 epitope to the antibody of the neurogenic acetylcholine receptor alpha 7-unit multi-strain is greater than the neurogenic acetylcholine receptor alpha 7-unit multi-drug antibody and the washing buffer The interaction between the two, so the neurogenic acetylcholine receptor alpha 7 units of multiple antibodies will not be washed away by the wash buffer.

加入200μL帶有辣根過氧化酶(Horseradish peroxidase,HRP)的羊抗兔IgG抗體(Sigma),25℃培養2小時。 200 μL of goat anti-rabbit IgG antibody (Sigma) with horseradish peroxidase (HRP) was added and cultured at 25 ° C for 2 hours.

以0.01M PBS清洗後,加入100μL 3,3',5,5'-四甲基聯苯胺(TMB;Invtrogen),室溫避光15~30分鐘。 After washing with 0.01 M PBS, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB; Invtrogen) was added, and the temperature was kept at room temperature for 15 to 30 minutes.

加入50μL 10% H2SO4終止反應,並檢測吸光值(OD值)。檢測波長為450nm,參考波長為630nm,須於10分鐘內完成檢測。 The reaction was terminated by the addition of 50 μL of 10% H 2 SO 4 and the absorbance (OD value) was measured. The detection wavelength is 450 nm, the reference wavelength is 630 nm, and the detection must be completed within 10 minutes.

將各組吸光值扣除負對照組之吸光值後,分別分析實驗抗原組1、2、3、4、5與負對照抗原組間是否均有顯著差異(p<0.01),正對照抗原組與負對照抗原組間是否有顯著差異(p<0.001)。 After subtracting the absorbance values of the negative control groups from each group, the experimental antigen groups 1, 2, 3, 4, 5 and the negative control antigen groups were analyzed for significant difference (p<0.01), and the positive control antigen group and There was a significant difference between the negative control antigen groups (p < 0.001).

神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子檢測精神分裂症效果分析:Neuroacridine choline receptor alpha 7-unit epitope linear peptide molecule detection of schizophrenia effect analysis:

A、樣本收集:A, sample collection:

收集306份精神分裂症患者血漿樣品及547例年齡與性別完全匹配的健康人血清樣本。精神分裂症的臨床診斷符合美國精神醫學會 DSM-IV診斷標準。 Plasma samples from 306 patients with schizophrenia and 547 healthy human serum samples with age and gender were matched. Clinical diagnosis of schizophrenia in line with the American Psychiatric Association DSM-IV diagnostic criteria.

B、檢測方式:B. Detection method:

利用上述免疫酵素測定法(ELISA)檢測精神分裂症病患自身抗體含量。檢測精神分裂症病患自身抗體之步驟、方法與上述神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子結合性分析之方法大致相同,步驟如下:首先,將本案SEQ ID NO:1、2、3、4、5神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子溶解後,以等比例混合。以0.01M PBS(pH7.0~7.4)將該神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子混合液之濃度調整成7.5~15μg/mL。較佳者,該0.01M PBS中包含0.1% NaN3The autoimmune assay (ELISA) was used to detect the autoantibody content of patients with schizophrenia. The method and method for detecting autoantibodies in a schizophrenia patient are substantially the same as the method for synthesizing a linear polypeptide molecule of an alpha 7-unit epitope in the above neurocholine acetylcholine receptor. The steps are as follows: First, the SEQ ID NO: 1, 2, 3, 4, 5 neuroacetylcholine receptors alpha 7 times unit epitope linear polypeptide molecules are dissolved, and mixed in equal proportions. The concentration of the linear acetylcholine receptor alpha-subunit epitope linear polypeptide molecule mixture was adjusted to 7.5-15 μg/mL in 0.01 M PBS (pH 7.0-7.4). Preferably, the 0.01 M PBS contains 0.1% NaN 3 .

以0.01M PBS(pH7.0~7.4)將正對照抗原及負對照抗原之濃度分別調整成15~20μg/mL。較佳者,該0.01M PBS中包含0.1% NaN3The concentrations of the positive control antigen and the negative control antigen were adjusted to 15-20 μg/mL in 0.01 M PBS (pH 7.0 to 7.4), respectively. Preferably, the 0.01 M PBS contains 0.1% NaN 3 .

將各種包被液分別包被(Coating)於多孔盤,每孔100μL,於4℃環境靜置隔夜(Overnight)。較佳者,該多孔盤為96孔盤,但不以此為限。以96孔盤(Cat.No.224-0096,COSTAR)為例,於A列的2個孔中包被神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子混合液,是為實驗抗原組。於B列的2個孔中包被正對照抗原包被液,是為正對照抗原組。於C列的2個孔中包被負對照抗原包被液,是為負對照抗原組。於D列的2個孔中包被0.01M PBS(pH7.0~7.4),是為負對照組。 Each of the coating liquids was individually coated on a porous disk, 100 μL per well, and allowed to stand overnight at 4 ° C (Overnight). Preferably, the porous disk is a 96-well disk, but is not limited thereto. Taking a 96-well plate (Cat. No. 224-0096, COSTAR) as an example, a mixture of a linear acetylcholine receptor alpha-subunit epitope linear polypeptide molecule is coated in two wells of column A, Experimental antigen group. A positive control antigen coating solution was coated in two wells of column B, which was a positive control antigen group. The negative control antigen coating solution was coated in two wells of column C, which was a negative control antigen group. 0.01 M PBS (pH 7.0 to 7.4) was coated in 2 wells of column D, which was a negative control group.

以0.01M PBS(另外添加0.05% TWEEN-20)清洗3遍。 Wash 3 times with 0.01 M PBS (additionally 0.05% TWEEN-20).

利用樣品稀釋分析液(0.01M PBS中添加1% BSA)將精神 分裂症病患之血漿稀釋100~500倍,每孔加入100μL,25℃培養2~3小時。 Use the sample dilution assay solution (1% BSA in 0.01M PBS) to apply the spirit The plasma of patients with schizophrenia is diluted 100-500 times, 100 μL per well, and cultured at 25 ° C for 2 to 3 hours.

以0.01M PBS(另外添加0.005% TWEEN-20)清洗3遍。加入200μL帶有辣根過氧化酶(Horseradish peroxidase,HRP)的羊抗人IgG抗體(Sigma),25℃培養2小時。 Wash 3 times with 0.01 M PBS (additionally 0.005% TWEEN-20). 200 μL of goat anti-human IgG antibody (Sigma) with horseradish peroxidase (HRP) was added and incubated at 25 ° C for 2 hours.

以0.01M PBS清洗後,加入100μL 3,3',5,5'-四甲基聯苯胺(TMB;Invtrogen),室溫避光15~30分鐘。 After washing with 0.01 M PBS, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB; Invtrogen) was added, and the temperature was kept at room temperature for 15 to 30 minutes.

加入50μL 10% H2SO4終止反應,並檢測吸光值(OD值)。檢測波長為450nm,參考波長為630nm。 The reaction was terminated by the addition of 50 μL of 10% H 2 SO 4 and the absorbance (OD value) was measured. The detection wavelength was 450 nm and the reference wavelength was 630 nm.

C、數據分析方式:C, data analysis method:

採用特異結合指數(Specific binding index,SBI)判定神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子與血漿自身抗體的結合程度。 The specific binding index (SBI) was used to determine the degree of binding of the linear 7-element epitope linear polypeptide molecule to plasma autoantibodies in the neuroacetylcholine receptor.

SBI=[實驗抗原組吸光值-負對照組吸光值]/[負對照抗原組吸光值-負對照組吸光值]。 SBI = [experimental antigen group absorbance - negative control absorbance] / [negative control antigen group absorbance - negative control absorbance].

採用Z檢定方法(Z test)分析精神分裂症病患血漿樣本的SBI值與健康人血漿樣本的SBI值之間是否有差異。 A Z-test was used to analyze whether there was a difference between the SBI value of plasma samples from patients with schizophrenia and the SBI values of healthy human plasma samples.

D、實驗結果:D. Experimental results:

請參閱表一。精神分裂症患者血漿中神經性乙醯膽鹼受器阿爾法7次單位IgG抗體明顯高於健康組,Z=6.27,具有統計差異(p<0.0001)。 Please refer to Table 1. The alpha 7-unit IgG antibody in the plasma of patients with schizophrenia was significantly higher than that of the healthy group, Z=6.27, with statistical difference (p<0.0001).

以上結果充分顯示,利用本案神經性乙醯膽鹼受器阿爾法7 次單位表位線性多肽分子所篩檢出的自身抗体含量,在精神分裂症患者與健康人之間具有顯著的統計差異。因此,本案神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子可作為臨床上檢測是否罹患精神分裂症的輔助性工具,以提供自體抗體數據作為客觀之診斷指標。 The above results fully show that the use of this case neuroic acetylcholine receptor alpha 7 The self-antibody content of the linear polypeptide molecules screened by the subunit epitope has significant statistical differences between patients with schizophrenia and healthy people. Therefore, the neuroic acetylcholine receptor alpha 7-element epitope linear polypeptide molecule can be used as an auxiliary tool for clinical detection of schizophrenia to provide autoantibody data as an objective diagnostic indicator.

神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子檢測準確性分析:Analysis of the accuracy of linear peptide detection of alpha 7-unit epitopes in neurogenic acetylcholine receptors:

A、檢測方式:以接收者操作特徵曲線(Receiver operating characteristic curve,ROC)評估本案神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子檢測效果。本分析之接收者操作特徵曲線,是依序設定不同閾值,並將SBI值高於閾值之個體的檢測結果定義為陽性,SBI值低於閾值之個體的檢測結果定義為陰性。 A. Detection method: The Receiver operating characteristic curve (ROC) was used to evaluate the linear peptide molecular detection effect of the 7-unit epitope of the neuroacetylcholine receptor in this case. The receiver operating characteristic curve of the analysis is to set different thresholds sequentially, and the detection result of the individual whose SBI value is higher than the threshold value is defined as positive, and the detection result of the individual whose SBI value is lower than the threshold value is defined as negative.

其中,精神分裂症患者檢測結果為陽性者,進一步定義為真陽性。精神分裂症患者檢測結果為陰性者,進一步定義為偽陰性。 Among them, those with schizophrenia test results are positive, further defined as true positive. Patients with schizophrenia whose test results are negative are further defined as pseudo-negative.

健康者檢測結果為陽性者,進一步定義為偽陽性。健康者檢測結果為陰性者,進一步定義為真陰性。 Those whose health test results are positive are further defined as false positives. Those whose health test results are negative are further defined as true negative.

統計每一種閾值所得出的真陽性比例(TPR)以及偽陽性比例(FPR)。例如,在第n個閾值下,統計出的真陽性比例為TPRn,偽陽性比例為FPRn。將(FPR1,TPR1)、(FPR2,TPR2)、(FPR3,TPR3)...等座標位置依序畫在接收者操作特徵曲線圖中。其中,接收者操作特徵曲線圖之橫座標為偽陽性比例(FPR),縱座標為真陽性比例(TPR)。發明人係使用Analyse-it for Microsoft Excel軟體繪製接收者操作特徵曲線。 The true positive ratio (TPR) and false positive ratio (FPR) obtained from each threshold were counted. For example, under the nth threshold, the true positive ratio is TPRn, and the false positive ratio is FPRn. The coordinate positions such as (FPR1, TPR1), (FPR2, TPR2), (FPR3, TPR3), etc. are sequentially drawn in the receiver operating characteristic graph. Among them, the abscissa of the receiver operating characteristic graph is a false positive ratio (FPR), and the ordinate is a true positive ratio (TPR). The inventor used the Analyse-it for Microsoft Excel software to plot the receiver operating characteristic curve.

將接收者操作特徵曲線進行積分,以求得接收者操作特徵曲線下面積(Area under the curve,AUC),進而能評估試驗之準確性。AUC數值越接近1.0者,代表診斷準確度越好,越接近0者,代表準確性越差。若AUC數值為1,表示檢測之準確性為100%。若AUC數值為0,表示檢測之準確性為0%。一般被市場採用之檢測方法,AUC均大於0.5。 The receiver operating characteristic curve is integrated to obtain the area under the curve (AUC) of the receiver, and the accuracy of the test can be evaluated. The closer the AUC value is to 1.0, the better the diagnostic accuracy is. The closer to 0, the worse the accuracy. If the AUC value is 1, the accuracy of the test is 100%. If the AUC value is 0, the accuracy of the detection is 0%. The detection method generally adopted by the market, the AUC is greater than 0.5.

B、實驗結果:B. Experimental results:

請參閱圖一以及表一。圖一係依精神分裂症群組及其匹配健康群組之SBI值所畫出的接收者操作特徵曲線。如表一所示,該ROC曲線下之面積為0.8,95%信賴區間為0.71~0.89。 Please refer to Figure 1 and Table 1. Figure 1 is a receiver operating characteristic curve drawn from the SBI values of the schizophrenia group and its matching healthy group. As shown in Table 1, the area under the ROC curve is 0.8, and the 95% confidence interval is 0.71 to 0.89.

以上結果顯示,利用本案神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子檢測是否罹患精神分裂症,接收者操作特徵曲線下面積不但大於0.5,且高達0.8。因此,本案之檢測方式具有相當的準確性及應用價值。 The above results show that the use of the linear acetylcholine receptor alpha alpha peptide linear polypeptide molecules in this case to detect schizophrenia, the area under the receiver operating characteristic curve is not only greater than 0.5, and up to 0.8. Therefore, the detection method of this case has considerable accuracy and application value.

神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子檢定方法之最適閾值:Optimal threshold for linear peptide molecular assays for alpha 7-unit epitopes in neuroketamine acetylcholine receptors:

依接收者操作特徵曲線上各個點的分布情形,選取最適之檢測閾值,使檢測方法具有高靈敏度(高靈敏度即真陽性比例高),同時具有高特異性(特異性高即真陰性比例高)。 According to the distribution of each point on the receiver's operating characteristic curve, the optimal detection threshold is selected to make the detection method have high sensitivity (high sensitivity, ie, high true positive ratio), and high specificity (high specificity, ie, true negative ratio) .

較佳者,本檢測方式之最適閾值為SBI=2,亦即個體之SBI值高於2時,檢測結果為陽性,可能罹患精神分裂症。反之,個體之SBI低 於2時,其檢測結果為陰性,該次檢測並未發現罹患精神分裂症。 Preferably, the optimal threshold of the detection method is SBI=2, that is, when the SBI value of the individual is higher than 2, the detection result is positive, and may be schizophrenia. Conversely, the individual's SBI is low. At 2 o'clock, the test result was negative, and the test did not find schizophrenia.

請參閱表一。以SBI=2作為閾值進行檢測時,精神分裂症之檢測靈敏度達21.9%,特異性達90.7%。 Please refer to Table 1. When SBI=2 was used as the threshold, the detection sensitivity of schizophrenia was 21.9% and the specificity was 90.7%.

應能理解的是,亦可選擇接收者操作特徵曲線上其他點所對應之閾值,作為區分陽性或陰性之閾值。 It should be understood that the threshold corresponding to other points on the receiver operating characteristic curve may also be selected as a threshold for distinguishing between positive and negative.

本發明所述的神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子可用於製備診斷試劑或免疫酵素測定套組(ELISA Kit),以檢測個體血液、血清或血漿中自身抗體含量,俾利診斷是否罹患一精神疾病。較佳者,用於檢測、診斷是否罹患精神分裂症。 The neuroic acetylcholine receptor alpha peptide unit linear polypeptide molecule of the invention can be used for preparing a diagnostic reagent or an immunozyme assay kit (ELISA Kit) for detecting autoantibody content in blood, serum or plasma of an individual. , Philip has diagnosed whether it is suffering from a mental illness. Preferably, it is used for detecting and diagnosing whether or not suffering from schizophrenia.

若檢測到的自身抗體含量升高,代表該個體之阿爾法7尼古丁受器之功能可能受阻斷/功能低下,亦即,個體可能罹患精神分裂症或神經性乙醯膽鹼受器阿爾法7次單位相關之其他精神疾病。 If the detected level of autoantibody is elevated, the function of the alpha 7 nicotine receptor representing the individual may be blocked/functioning, ie, the individual may have schizophrenia or neurocholine acetylcholine receptor 7 times. Other mental illnesses related to the unit.

利用本發明之神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子製備之免疫酵素測定套組,包含一多孔盤,且該多孔盤中包被有SEQ ID NO:1、2、3、4、或5之神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子。較佳者,該多孔盤係96孔盤。較佳者,該免疫酵素測定套組更包括選自下列各表之試劑: An immunoenzyme assay kit prepared by using the alpha 7-element epitope linear polypeptide molecule of the neurogenic acetylcholine receptor of the present invention, comprising a porous disk, and the porous disk is coated with SEQ ID NO: 1, 2 , 3, 4, or 5 neuroacetylcholine receptor alpha alpha subunit epitope linear polypeptide molecule. Preferably, the porous disk is a 96-well disk. Preferably, the immunoenzyme assay kit further comprises reagents selected from the following tables:

利用本發明之神經性乙醯膽鹼受器阿爾法7次單位表位線性多肽分子所製備之精神疾病診斷試劑或免疫酵素測定套組,可檢測個體血液、血漿、或血清中的α 7 nAChR自身抗體含量,進而作為臨床醫生綜合診斷精神疾病時之客觀依據。 The psychiatric diagnostic reagent or immunoenzyme assay kit prepared by using the linear 7-element epitope linear polypeptide molecule of the neurogenic acetylcholine receptor of the present invention can detect α 7 nAChR itself in blood, plasma, or serum of an individual. The antibody content is used as an objective basis for clinicians to comprehensively diagnose mental illness.

以上所述僅為本發明之較佳實施例,並非用以限定本發明之申請專利範圍,因此凡其它未脫離本發明所揭示之精神下所完成之各種更動或潤飾等,均應包含於本案之申請專利範圍內。 The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the claims of the present invention. Therefore, various other modifications or retouchings, etc., which are not departing from the spirit of the present invention, should be included in the present invention. Within the scope of the patent application.

以下序列的胺基酸係由左到右以胺基到羧基的方向表示。 The amino acid of the following sequence is represented from the left to the right in the direction of the amine group to the carboxyl group.

<110> 光輝生命醫學股份有限公司 <110> Guanghui Life Medicine Co., Ltd.

<120> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體之多肽分子 <120> A polypeptide molecule for detecting an alpha subunit antibody of a neurocholine acetylcholine receptor

<160> 5 <160> 5

<210> 1 <210> 1

<211> 33 <211> 33

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> PEPTIDE <220> PEPTIDE

<223> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體 <223> For detecting neurogenic acetylcholine receptor alpha subunit antibody

<400> 1 <400> 1

<210> 2 <210> 2

<211> 33 <211> 33

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> PEPTIDE <220> PEPTIDE

<223> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體 <223> For detecting neurogenic acetylcholine receptor alpha subunit antibody

<400> 2 <400> 2

<210> 3 <210> 3

<211> 26 <211> 26

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> PEPTIDE <220> PEPTIDE

<223> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體 <223> For detecting neurogenic acetylcholine receptor alpha subunit antibody

<400> 3 <400> 3

<210> 4 <210> 4

<211> 29 <211> 29

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> PEPTIDE <220> PEPTIDE

<223> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體 <223> For detecting neurogenic acetylcholine receptor alpha subunit antibody

<400> 4 <400> 4

<210> 5 <210> 5

<211> 27 <211> 27

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> PEPTIDE <220> PEPTIDE

<223> 用於檢測神經性乙醯膽鹼受器阿爾法次單位抗體 <223> For detecting neurogenic acetylcholine receptor alpha subunit antibody

<400> 5 <400> 5

Claims (13)

一種抗體結合分子,可與一神經性乙醯膽鹼受器阿爾法7次單位(Neuronal acetylcholine receptor subunit alpha-7)之一抗體結合;其中,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 An antibody binding molecule that binds to an antibody of neuronal acetylcholine receptor subunit alpha-7; wherein the antibody binding molecule is 25-40 amino acids in length And the sequence of the antibody binding molecule comprises at least one of SEQ ID NOS: 1 to 5. 如申請專利範圍第1項所述之抗體結合分子,其序列長度為25至35個胺基酸。 The antibody-binding molecule of claim 1, which has a sequence length of 25 to 35 amino acids. 如申請專利範圍第2項所述之抗體結合分子,其序列中的脯胺酸(Proline)數量為0至3個。 The antibody binding molecule of claim 2, wherein the number of proline in the sequence is from 0 to 3. 如申請專利範圍第2項所述之抗體結合分子,其序列中的麩胺酸(Glutamic acid)數量為0至3個,抑或是,其序列中的半胱胺酸(Cysteine)數量為0至1個。 The antibody-binding molecule according to claim 2, wherein the number of glutamic acid (Glutamic acid) in the sequence is 0 to 3, or the number of Cysteine in the sequence is 0 to One. 如申請專利範圍第2項所述之抗體結合分子,係線性多肽分子,抑或是,該抗體結合分子於一水溶液或一血漿中不形成阿爾法螺旋(alpha helix)結構。 The antibody-binding molecule according to claim 2 is a linear polypeptide molecule, or the antibody-binding molecule does not form an alpha helix structure in an aqueous solution or a plasma. 如申請專利範圍第2項所述之抗體結合分子,其與該抗體間的結合力,大於該抗體與一洗滌緩衝液間的作用力。 The antibody binding molecule according to claim 2, wherein the binding force to the antibody is greater than the interaction between the antibody and a washing buffer. 一種檢測試劑,包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 A detection reagent comprising an antibody binding molecule having a length of 25-40 amino acids, and the sequence of the antibody binding molecule comprises at least one of SEQ ID NOS: 1 to 5. 一種免疫酵素測定套組(ELISA Kit),包含一試劑;該試劑中更包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 An immunoassay kit (ELISA Kit) comprising a reagent; the reagent further comprises an antibody binding molecule having a length of 25-40 amino acids, and the sequence of the antibody binding molecule comprises SEQ ID NO: at least one of 1 to 5. 如申請專利範圍第8項所述之免疫酵素測定套組,該試劑中之該抗體結合分子濃度為0.75μg/mL~5mg/mL。 The immunoenzyme assay kit according to item 8 of the patent application, wherein the antibody binding molecule concentration in the reagent is 0.75 μg/mL to 5 mg/mL. 一種免疫酵素測定套組(ELISA Kit),包含一多孔盤;該多孔盤中包含一抗體結合分子,該抗體結合分子之長度為25-40個胺基酸,且該抗體結合分子之序列包含SEQ ID NO:1至5之至少一者。 An immunoassay kit (ELISA Kit) comprising a porous disc; the porous disc comprising an antibody binding molecule having a length of 25-40 amino acids, and the sequence of the antibody binding molecule comprises At least one of SEQ ID NOS: 1 to 5. 如申請專利範圍第8或10項所述之免疫酵素測定套組,係用於檢測一血液,一血漿,或一血清中的一神經性乙醯膽鹼受器阿爾法7次單位之抗體含量。 The immunoenzyme assay kit described in claim 8 or 10 is for detecting the antibody content of an alpha 7-unit unit of a neurotransmitter choline receptor in a blood, a plasma, or a serum. 如申請專利範圍第8或10項所述之免疫酵素測定套組,可用於早期診斷是否罹患一精神疾病或作為追蹤一精神疾病病況之指標。 The immunoenzyme assay kit described in claim 8 or 10 can be used for early diagnosis of a mental illness or as an indicator for tracking a psychiatric condition. 如申請專利範圍第12項所述之免疫酵素測定套組,其中該精神疾病係精神分裂症。 The immunoenzyme assay kit of claim 12, wherein the mental illness is schizophrenia.
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