CN116559465A - 抗ncam1自身抗体的试剂在制备检测神经系统症状相关疾病的产品中的应用及试剂盒 - Google Patents
抗ncam1自身抗体的试剂在制备检测神经系统症状相关疾病的产品中的应用及试剂盒 Download PDFInfo
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Abstract
本发明公开了抗NCAM1自身抗体的试剂在制备检测神经系统症状相关疾病的产品中的应用及试剂盒,属于生物医药技术领域。本发明首次以检测抗NCAM1自身抗体的试剂为主要成分,建立检测神经系统相关疾病的试剂盒。利用本发明试剂盒能够定性或定量分析抗NCAM1自身抗体,操作简便,辅助检测和/或诊断神经系统相关疾病。
Description
技术领域
本发明属于生物医药技术领域,具体涉及抗NCAM1自身抗体的试剂在制备检测神经系统症状相关疾病的产品中的应用及试剂盒。
背景技术
神经细胞粘附分子1(Neural Cell Adhesion Molecule1,NCAM1),又称CD56或MSK39,是免疫球蛋白超家族中的一员,也是神经细胞黏附分子中的一员,主要表达于中枢和外周神经系统、免疫系统、甲状腺、肾上腺、心脏、肾脏和胃,其主要生物学功能是参与细胞发育和分化过程中细胞-细胞和细胞-基质的的粘附和信号传导,调节神经细胞功能,在神经细胞迁移起关键作用。人NCAM1基因定位于11q22-23,是一种I型膜糖蛋白,有多种选择性剪切产生的亚型,NCAM1常见的三种蛋白亚型在变性凝胶电泳条件下迁移的表观分子质量分别为120、140和180kDa,因此分别命名为NCAM-120、NCAM-140和NCAM-180,其中NCAM-120是正常成人肾脏组织中最常见的亚型,NCAM-140是胚胎发育、CD56+恶性肿瘤和肿瘤细胞系中的显著亚型,NCAM1在多种原发和转移性肿瘤,如肺癌、多发性骨髓瘤、Merkel细胞癌、卵巢癌和脑胶质瘤等中高表达,参与细胞分化、迁移和侵袭以及影响肿瘤的转移性扩散。NCAM1是肺癌的重要标记物和潜在靶点,NCAM1在几乎绝大多数小细胞肺癌细胞膜表面特异性高表达,是肺癌抗体治疗和抗体偶联药物治疗的理想靶点,同时NCAM1在一些免疫细胞和神经来源的细胞表面表达,因此NCAM1也可能作为一些神经系统自身免疫疾病的治疗靶点。NCAM1在癌症发病机制中的作用已被广泛描述,然而,它可能作为抗原参与副肿瘤神经病变或脑炎病变尚未被研究。
神经系统自身免疫性疾病是神经病学领域中的一大类重要疾病,神经系统自身免疫性疾病是以自身免疫细胞、免疫分子等攻击神经系统为主要致病机制的自身免疫性疾病,具有免疫疾病的复杂性和神经系统疾病的高致死、致残性的特点,因而受到临床医师和研究者的高度关注。神经系统自身免疫性疾病可发生在中枢神经系统、周围神经系统及神经-肌肉接头处,常见的神经系统自身免疫性疾病包括:自身免疫性脑炎(autoimmuneencephalitis,AE)、中枢神经系统脱髓鞘疾病、僵人综合征(stiff person syndrome,SPS)、免疫介导性周围神经病、自身免疫性小脑共济失调、肌无力综合征等。
在免疫反应中,作用于神经系统自身抗原的致病抗体统称为神经系统自身抗体。近年来,随着对神经系统自身抗体认知的扩展及检测技术的进步,越来越多的神经系统自身免疫性疾病被确诊。目前针对抗NCAM1自身抗体在神经系统副肿瘤综合征或自身免疫性脑炎患者中的存在情况未见报道。
发明内容
本发明的目的在于提供一种抗NCAM1自身抗体的试剂在制备检测神经系统症状相关疾病的产品中的应用及试剂盒。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种自身免疫性疾病标志物在制备用于神经系统相关疾病诊断的检测试剂或检测试剂盒中的应用,所述自身免疫性疾病标志物为与NCAM1蛋白结合的自身抗体,在检测时是检测样品与NCAM1蛋白结合的自身抗体。
优选地,所述NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
进一步优选地,编码SEQ ID NO.1所示的氨基酸序列的核苷酸序列包括Ⅰ)~Ⅲ)中的任意一种:
Ⅰ)SEQ ID NO.2所示的核苷酸序列;
Ⅱ)SEQ ID NO.2所示的核苷酸序列的10%~80%,且编码识别NCAM1自身抗体的氨基酸序列;
Ⅲ)Ⅰ)或Ⅱ)中的核苷酸序列突变后,且编码识别NCAM1自身抗体的氨基酸序列。
优选地,所述神经系统相关疾病包括副肿瘤综合征、小脑变性和边缘性脑炎。
进一步优选地,所述神经系统相关疾病包括头痛,眩晕、恶心、呕吐,复视,步态肢体共济失调,构音障碍,眼球震颤,记忆力障碍,智能减退、近端肌无力或四肢肌肉缺乏协调性的症状。
优选地,检测样品为全血、血清和脑脊液的一种或几种。
本发明还公开了一种检测自身免疫性疾病的蛋白在制备用于神经系统相关疾病诊断的检测试剂或检测试剂盒中的应用,所述检测自身免疫性疾病的蛋白为衍生自NCAM1蛋白的一个或多个表位,或者为融合其他氨基酸的融合蛋白;其中,NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
本发明还公开了一种检测神经系统相关疾病的试剂盒,包括检测抗NCAM1抗体的试剂和标记抗体;其中:
所述检测抗NCAM1抗体的试剂包括NCAM1蛋白、表达NCAM1蛋白的细胞、表达NCAM1蛋白的组织和含有NCAM1蛋白的裂解物中的一种或多种;
所述标记抗体为能够与人IgG的Fc片段结合的抗体。
优选地,所述NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
优选地,所述标记抗体包括辣根过氧化物酶标记抗人抗体、碱性磷酸酶标记抗人抗体、生物素标记的抗人抗体、FITC标记的抗人抗体或Alexa Fluor染料标记的抗人抗体。
与现有技术相比,本发明具有以下有益效果:
本发明提供了抗NCAM1自身抗体的试剂在制备诊断神经系统症状相关疾病的试剂盒中的应用,本发明首次以检测抗NCAM1抗体的试剂为主要成分,建立检测神经系统相关疾病的试剂盒。利用本发明试剂盒能够定性或定量分析抗NCAM1抗体,操作简便,辅助检测和/或诊断神经系统相关疾病。
本发明提供了一种检测神经系统相关疾病的试剂盒,包括检测抗NCAM1抗体的试剂和标记抗体,该试剂盒能够实现NCAM1自身抗体的检测,并且可以用来诊断神经系统疾病。
进一步地,本申请的试剂盒能够检测一种或多种症状相关的神经系统疾病,所述症状包括头痛,眩晕、恶心、呕吐,复视,步态肢体共济失调,构音障碍,眼球震颤,记忆力障碍,智能减退,近端肌无力,四肢肌肉缺乏协调性。本发明所述应用制备得到的试剂盒还可以用来区分自身免疫疾病,尤其是区分神经系统的自身免疫疾病与非自身免疫疾病。为疾病的诊疗提供依据,有利于确定最有希望的治疗方案。
附图说明
图1为实施例1中患者1、患者2和正常对照血清在原代细胞上的染色结果;
图2为实施例1中患者1、患者2血清与神经元marker Tubb3抗体共染结果;
图3为实施例2中患者1血清和对照血清在大鼠脑组织冰冻切片上染色结果;
图4为实施例3中患者1和正常对照血清在过表达NCAM1细胞爬片染色结果;
图5为实施例3中患者1血清与NCAM1抗体共染结果;
图6为实施例4中NCAM1抗体在WB上验证过表达蛋白结果;
图7为实施例4中血清中和实验在CBA上验证患者血清所检信号;
图8为实施例4中NCAM1抗体在WB上验证鼠脑组织蛋白结果;
图9为实施例4中血清中和实验在鼠脑组织切片上验证患者血清所检信号;
图10为实施例4中血清中和实验在免疫印迹上验证患者血清所检信号;
图11为实施例5中回收自身抗体在过表达细胞爬片上染色结果;
图12为实施例5中回收自身抗体在鼠脑组织切片上染色结果;
图13为实施例6中筛选出9例抗NCAM1自身抗体阳性患者在过表达NCAM1细胞爬片的染色结果。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
本发明实施例中的患者血清均为医院赠予,已经经过本人同意,健康受试者血清来自于医院体检中心赠送的健康体检者血清,已经体检者本人同意,患者信息如下:
患者1:女,65岁,患者失眠,眩晕,复视,近期伴有呕吐,肢体颤抖,同时伴有记忆力减退,语言障碍等症状故到医院就诊。查体发现该患者眼球震颤,指鼻不准,医生怀疑其患有神经系统自身免疫性疾病,将其样本送往检验科,经检测,该样本副肿瘤14项(包括Ri、Hu、Yo、CV2、Ma2、Amphiphysin、Titin、Ma1、SOX1、Tr、Zic4、PKCγ、Recoverin、GAD65)阴性,自免性脑炎15项(包括NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin3α)阴性,中枢脱髓鞘病变6项(AQP4、MBP、MOG、GFAP、AQP1、Flotillin1/2)阴性,外周神经病1项(GQ1b IgG和GQ1b IgM)阴性。
患者2:男,78岁,表现为头痛,记忆力差,智能减退,近事记忆模糊,医生对其进行既往病史询问时,不能清楚自述病情,需家属帮助,做颅脑MRI显示双侧海马区脱髓鞘改变,考虑脑病,予激素及阿昔洛韦抗病毒治疗病情有好转,60d后再进一步加重,智能进行性减退,并出现肌无力综合征,医生怀疑其患有神经系统自身免疫性疾病,将其样本送往检验科,经检测,该样本副肿瘤14项(包括Ri、Hu、Yo、CV2、Ma2、Amphiphysin、Titin、Ma1、SOX1、Tr、Zic4、PKCγ、Recoverin、GAD65,印迹法)阴性,自免性脑炎15项(包括NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin3α,免疫荧光法)阴性,中枢脱髓鞘病变6项(AQP4、MBP、MOG、GFAP、AQP1、Flotillin1/2,免疫荧光法)阴性,外周神经病1项(GQ1b IgG和GQ1b IgM,印迹法)阴性。
实施例1免疫荧光法检测患者样本在大鼠脑组织原代细胞的荧光信号
通过大鼠脑组织原代细胞免疫荧光实验的探究,发现上述患者的血清中筛选出可与大鼠脑组织原代细胞发生免疫反应的自身抗体。具体实验步骤如下:
步骤1、大鼠脑组织原代细胞的分离
10%水合氯醛麻醉孕鼠,75%酒精浸泡消毒3min,生物安全柜取出胎鼠;使用预冷的Hanks(KCl:0.4g/L,NaCl:8g/L,NaHCO3:0.35g/L,Na2HPO4:0.0477g/L,葡萄糖:1g/L,Hepes:5g/L,溶剂为水)冲洗胎鼠两次,在预冷的HBSS中解剖出胎脑浸泡在预冷的DMEM中;弃DMEM,用眼科剪将组织块剪成糜状混合物。加入木瓜蛋白酶,37℃消化处理30分钟;消化结束后,将细胞移到新的离心管,加入DMEM重悬,400g、4℃离心5min,弃上清液,再次加入DMEM重悬,400g、4℃离心5min,弃上清液,加入20ml Neurobasal(厂家:GIBCO货号:21103-049)培养基重悬细胞。后续实验使用OptiPrep介质(厂家:sigma货号:92339-11-2)将上述重选细胞中分离出原代细胞,置于放有玻片的细胞培养皿中培养。将大鼠原代细胞爬片经4%多聚甲醛(PFA)固定10min,HEPES洗2遍,1.25M的甘氨酸终止10min,HEPES洗2遍,得到爬片。
步骤2、血清孵育
使用PBS将患者1、患者2和正常对照血清按1:50进行稀释,分别孵育至步骤1制备的大鼠脑组织原代细胞爬片上,室温孵育1h,HEPES洗2次,每次5min;再加入稀释好的FITC标记的羊抗人IgG(厂家:Jackson货号:109-095-170),室温孵育30min,HEPES洗2次,每次5min;DAPI对细胞核进行染色,室温染色10min,HEPES洗2次,每次5min;显微镜下观察,结果如图1所示。根据图1可以看出患者1、患者2血清在大鼠脑组织原代细胞爬片上出现阳性信号而正常对照血清在大鼠脑组织原代细胞爬片上未出现阳性信号,提示患者1、患者2血清中可能存在识别原代神经元细胞抗原的抗体。
步骤3、抗体共染
使用神经元特异性marker Tubb3抗体(厂家:武汉三鹰货号:66375-1-Ig)对步骤2孵育过患者1、患者2血清的爬片进行抗体共染,Tubb3抗体1:200稀释,HEPES洗2次,每次5min;Alexa Fluor 594标记的羊抗鼠IgG(厂家:Jackson货号:115-585-146)室温孵育30min,HEPES洗2次,每次5min;显微镜下观察,结果如图2所示。根据图2可以观察到,患者1、患者2血清染出的信号与Tubb3抗体在原代细胞神经元上的信号重叠,(Tubb3蛋白主要在神经元中表达)表明患者1、患者2血清中抗体所识别的抗原存在于神经元细胞上。
实施例2免疫荧光法检测患者样本在大鼠脑组织切片上的荧光信号
通过大鼠脑组织切片的免疫荧光实验的探究,发现上述患者的血清中存在可与大鼠脑组织切片发生免疫反应的自身抗体。具体实验步骤如下:
步骤1、大鼠脑组织冰冻切片的制备
选取成年大鼠进行麻醉,待老鼠四肢硬化后打开腹腔,暴露出心尖,从左心尖灌注PBS,以便全身循环;然后取出脑组织,甲醇固定10~30min;将样本移入30%蔗糖溶液中进行脱水,4℃放置至组织块沉底;将少量包埋剂OCT滴加到标本台,置入-20℃冷冻切片机(厂家:LEICA型号:CM1950)的冷冻台,待组织略微发白时用OCT在标本表面涂一薄层,继续冷冻20min,进行切片。
步骤2、血清孵育
使用PBST将患者1和正常对照血清按1:10进行稀释,孵育至大鼠脑组织冰冻切片上,室温孵育1h,PBST洗3次,每次5min;加入稀释好的FITC标记的羊抗人二抗(厂家:Jackson货号:109-095-170),室温孵育30min,PBST洗3次,每次5min,荧光显微镜下观察结果,结果如图3所示。根据图3可以看出患者1血清在大鼠脑组织冰冻切片上海马和皮层部位出现阳性信号而正常对照血清在此部位未出现阳性信号,提示患者1血清中可能存与大鼠脑组织中海马和皮层部位抗原相结合的抗体。
实施例3目标抗原的筛选与鉴定
根据送检信息得知患者1和患者2中枢脱髓鞘6项自身抗体检测、自身免疫性脑炎15项自身抗体检测、副肿瘤14项自身抗体检测及外周神经病相关疾病1项自身抗体检测共26项自身抗体检测均为阴性,但根据实施例1和实施例2中的染色结果可知,患者1和患者2血清中存在识别神经元细胞抗原的抗体。因此,初步判定患者1和患者2样本中存在不同于以往报道的新的自身抗体。
本实施例从https://www.proteinatlas.org/网站中查找在人大脑海马和皮层(神经元细胞)部位表达的100种蛋白名称(现有文献并未报道这些蛋白为神经系统自身免疫疾病的自身抗体所识别),在NCBI上查找编码所述100种蛋白的基因序列并送往测序公司进行基因合成,获得重组载体,然后将重组载体转染至293T细胞获得重组细胞,制备成检测用细胞爬片,最后通过免疫荧光法检测患者血清/脑脊液是否能与该细胞孵育后发生免疫反应,探索患者血清/脑脊液中是否含有该系列基因的特异性自身抗体,具体如下:
步骤1、重组载体构建
将上述100种在脑组织高表达基因通过分子克隆方法分别合成至pCDNA3.1载体上,得到对应基因的重组载体,将各重组载体分别转化至100支TOP-10感受体细胞中进行扩增,构建好的重组载体经质粒小提测序正确后大提备用;
步骤2、细胞转染
使用10%FBS-DMEM高糖培养基于37℃,5%CO2细胞培养箱中培养皿底铺有6cm×6cm爬片的293T细胞,共计101皿,待细胞密度达到30%~40%时,使用转染试剂PEI将100种对应基因的重组载体和空载pCDNA3.1分别转染至293T细胞中,并进行标记;
步骤3、细胞爬片固定
将转染过后生长48h的101皿细胞用PBS洗涤2次,加入丙酮固定5min,丙酮固定后的爬片用PBS洗涤2次,制成对应基因的过表达细胞爬片和空载对照细胞爬片干燥,将干燥后的细胞爬片裁成2.5mm×2.5mm大小的爬片,并将101种2.5mm×2.5mm的细胞爬片贴在载玻片上,备用;
步骤4、免疫荧光染色
一抗孵育:使用PBST将患者1血清和对照受试者血清分别按照1:10的比例稀释后孵育至过表达细胞爬片上,室温孵育1h,PBST洗3次,每次5min;
二抗孵育:使用FITC标记的抗人二抗孵育过表达细胞爬片,室温孵育30min;PBST洗3次,每次5min;荧光显微镜下观察。
结果患者1血清与上述100种脑组织抗原中的任意一种抗原均未产生区别于对照血清的信号,但根据实施例1和实施例2中的染色结果可知,患者1血清中存在识别神经元细胞抗原的抗体,故重新进行目的抗原的扩大筛选。方法同上述步骤,重新查找另外100种在人大脑海马和皮层(神经元细胞)部位表达的蛋白。从https://www.proteinatlas.org/网站中重新查找在人大脑海马和皮层(神经元细胞)部位表达的另外100种蛋白名称(现有文献并未报道这些蛋白为神经系统自身免疫疾病的自身抗体所识别),在NCBI上查找编码所述100种蛋白的基因序列(包括NCAM-180序列)并送往测序公司进行基因合成,获得重组载体,然后将重组载体转染至293T细胞获得重组细胞,制备成检测用细胞爬片,最后通过免疫荧光法检测患者血清/脑脊液是否能与该细胞孵育后发生免疫反应,探索患者血清/脑脊液中是否含有该系列基因的特异性自身抗体。
通过第二次目标抗原的筛选,结果患者1血清与二次筛选的100种脑组织蛋白的过表达细胞爬片中的一种细胞爬片发生反应,产生了阳性信号,而对照血清与二次筛选的100种蛋白的过表达细胞爬片均未发生反应,患者1血清与对照受试者血清的检测结果如图4所示。经查证,该蛋白为NCAM1(Neural Cell Adhesion Molecule 1神经细胞黏附分子1)。
步骤5、抗体共染
为进一步验证该目的抗原为NCAM1,选用商业化NCAM1抗体(厂家:武汉三鹰,货号:14255-1-AP)与患者1血清孵育过的过表达NCAM1细胞爬片进行抗体共染,NCAM1抗体1:200稀释,室温孵育1h,PBST洗3次,每次5min;使用Alexa Fluor 594标记的羊抗兔IgG(厂家:Jackson货号:115-585-144)室温孵育30min,PBST洗3次,每次5min;荧光显微镜下观察,共染结果如图5所示。
抗体共染结果显示,患者1在过表达NCAM1细胞爬片上染出的信号与NCAM1抗体染出的信号重叠,说明患者1血清中抗体与过表达细胞上的NCAM1蛋白特异性识别。
实施例4血清中和实验验证患者样本所检信号
步骤1、中和蛋白的制备
根据实施例3步骤2收集1皿过表达NCAM1的293T细胞和空载pCDNA3.1细胞,室温800rpm离心去上清,加入200μL PBS,超声破碎(破碎条件为:10%功率,破碎3s,停6s,共超声1min),作为NCAM1中和蛋白;以转染空载pCDNA3.1的细胞制备对照蛋白,制备条件与方法与NCAM1中和蛋白的制备相同;
步骤2、中和蛋白的鉴定
将收获的NCAM1中和蛋白和对照蛋白作为上样样品,测定浓度后各取40μg蛋白进行SDS-PAGE凝胶电泳,电泳结束后采用转膜条件为300mA,90min进行湿法转膜;5%的脱脂奶粉室温封闭1h;使用TBST将NCAM1抗体进行1:1000稀释,4℃孵育过夜;次日,TBST洗3次,每次5min;加入HRP标记的羊抗T兔二抗(厂家:Jackson),室温孵育1h;TBST洗3次,每次5min;加入化学发光液显色拍照,结果如图6所示。
步骤3、血清中和实验在过表达NCAM1细胞爬片上验证患者血清所检信号
使用PBST共制备3份1:10稀释的患者1血清,每份100μL,分别加入20μL PBST、20μLNCAM1中和蛋白和20μL对照中和蛋白室温孵育30min,分别用其孵育制备好的过表达NCAM1的细胞爬片,室温孵育1h,PBST洗3次,每次5min;加入1:200稀释的FITC标记的羊抗人二抗(厂家:Jackson货号:109-095-170),室温孵育30min,PBST洗3次,每次5min,荧光显微镜下观察,结果如图7所示。
步骤4、血清中和实验在鼠脑组织切片上验证患者血清所检信号
(1)Western Blot鉴定大鼠脑组织中NCAM1蛋白
大鼠脑组织各取50mg分别剪碎,转移至研钵中加入液氮冷冻研磨至细腻粉末状,各组样本分别加入500μLRIPA裂解液(150mM NaCl,1mM EDTA,100mM Tris-HCl,0.1%SDS,0.5%脱氧胆酸钠,1% TritonX-100,5%甘油,pH7.5),加入终浓度为1×的蛋白酶抑制剂,超声:10%功率,超3sec停6sec,总计1min。超声后冰上裂解30min,间隔5min震荡一次,15000rpm离心30min,取上清用BCA法测定蛋白浓度,取20μg样品进行上样。
电泳结束后采用转膜条件为30mA,90min进行湿法转膜;5%的脱脂奶粉室温封闭1h,以1:2000稀释的NCAM1抗体作为一抗4℃过夜孵育,次日,TBST洗3次,每次5min;然后加入HRP标记的羊抗兔二抗(厂家:Jackson)孵育1h;TBST洗3次,每次5min;加入ECL化学发光液显色拍照。实验结果如图8所示。
(2)血清中和实验
使用PBST共制备3份1:10稀释的患者血清,每份100μL,分别加入20μLPBST、20μLNCAM1中和蛋白和20μL对照中和蛋白,室温孵育30min,分别用其孵育制备好的大鼠脑组织冰冻切片,室温孵育1h,PBST洗3次,每次5min;加入1:200稀释的FITC标记的羊抗人二抗(厂家:Jackson货号:109-095-170),室温孵育30min,PBST洗3次,每次5min,荧光显微镜下观察,结果如图9所示。
步骤5、血清中和实验在免疫印迹上验证患者血清所检信号
(1)SDS-PAGE蛋白样品的制备
根据实施例3步骤2收集1皿过表达NCAM1的293T细胞和空载pCDNA3.1细胞,室温800rpm离心去上清,加入200μL PBS,超声破碎(破碎条件为:10%功率,破碎3s,停6s,共超声1min)15000rpm离心30min,取上清用BCA法测定蛋白浓度。
(2)上样及转膜
分别取40μg过表达NCAM1蛋白和pCDNA3.1蛋白进行上样,共制备3组,电泳结束后采用转膜条件为300mA,90min进行湿法转膜;5%的脱脂奶粉室温封闭1h;
(3)血清中和实验
使用PBST共制备3份1:300稀释的患者血清,每份1500μL,分别加入10μL PBST、10μL NCAM1中和蛋白和10μL对照中和蛋白,室温孵育30min,再将其各自孵育至转膜后的PVDF膜上,4℃孵育过夜;次日,TBST洗3次,每次5min;加入HRP标记的羊抗人二抗(厂家:Jackson),室温孵育1h;TBST洗3次,每次5min;加入ECL化学发光液显色拍照,GAPDH为内参蛋白,实验结果如图10所示。
结果分析:由图6可以看出,对比实验结果可发现,过表达NCAM1蛋白显色明显强于对照蛋白,因此认为NCAM1蛋白成功在293T细胞上过表达;由图7可以看出,在过表达NCAM1细胞爬片上,患者血清信号被NCAM1中和蛋白封闭,而对照蛋白未封闭住过表达NCAM1细胞爬片上出现的信号,表明该信号为特异性识别NCAM1抗原的信号;由图8可以看出,大鼠脑组织上有NCAM1蛋白的表达;由图9可以看出,在鼠脑切片上,患者血清信号被NCAM1中和蛋白封闭,而对照蛋白未封闭住组织切片上出现的信号,表明该信号为鼠脑组织上表达的NCAM1抗原的特异性信号。由图10可以看出,在免疫印迹上,患者血清被NCAM1中和蛋白封闭,而对照蛋白未封闭住患者血清在免疫印迹上出现的信号,表明该信号为特异识别NCAM1抗原的信号。内参蛋白GAPDH的显色可以说明过表达NCAM1蛋白和空载pCDNA3.1蛋白的上样量一致。
实施例5回收患者血清中自身抗体实验验证患者样本所检信号
验证患者血清中的自身抗体可特异识别NCAM1蛋白,并且制备一份与患者血清为相对阴性的血清,提前将稀释的患者血清分为两份,分别用其孵育过表达NCAM1细胞爬片和pCDNA3.1细胞爬片,采用甘氨酸洗脱、Tris中和的方法,将与过表达NCAM1细胞爬片和pCDNA3.1细胞爬片结合的血清中的有效成分洗脱下来,超滤管进行超滤浓缩,收获一份回收自身抗体洗脱液和对照洗脱液,理论上与对照洗脱液相比,自身抗体洗脱液为可与过表达NCAM1细胞爬片特异性结合的抗体洗脱液,包含非特异性结合成分,而对照洗脱液仅为与pCDNA3.1细胞爬片非特异结合的洗脱液,故可将对照洗脱液认为是自身抗体洗脱液的阴性对照样品,具体步骤如下:
步骤1、患者血清中自身抗体回收
参考实施例3在10cm细胞培养皿中分别培养过表达NCAM1细胞和空载pCDNA3.1细胞各一皿,并在培养皿里进行细胞固定,取200μL患者1血清用PBST按照1:50稀释,得到10mL稀释液,各取5mL稀释液加入到上述已进行细胞固定的培养皿中,4℃孵育过夜,次日,弃血清稀释液,向培养皿中加入PBS轻柔晃动洗涤5次,每次5min;洗涤结束后,每皿加入3mL pH=3的0.1M甘氨酸洗脱液,摇晃培养皿使洗脱充分,洗脱时间为15min,洗脱结束后向培养皿中加入100μL 1M的Tris中和至洗脱液pH值为7.0-8.0时,使用50kD超滤管(厂家:merck货号:UFC8050)对洗脱液进行超滤浓缩15倍,分别得到200μL含抗体的洗脱液和对照洗脱液备用;
步骤2、回收自身抗体在过表达细胞爬片上验证患者样本所检信号
取步骤1中回收的患者1含抗体的洗脱液100μL和对照洗脱液100μL,分孵育至过表达NCAM1的细胞爬片上,室温孵育1h,PBST洗3次,每次5min;使用FITC标记的抗人二抗孵育过表达细胞爬片,室温孵育30min;PBST洗3次,每次5min;荧光显微镜下观察,结果如图11所示。
步骤3、回收自身抗体在鼠脑组织切片上验证患者样本所检信号
取步骤1中回收的患者1含抗体的洗脱液100μL和对照洗脱液100μL,分别孵育至鼠脑组织切片上,室温孵育1h,PBST洗3次,每次5min;使用FITC标记的抗人二抗孵育过表达细胞爬片,室温孵育30min;PBST洗3次,每次5min;荧光显微镜下观察,结果如图12所示。
结果分析:由图11可以看出,回收的患者1自身抗体的洗脱液在过表达NCAM1细胞爬片上有阳性信号,而在空载pCDNA3.1细胞爬片上无阳性信号,回收的对照洗脱液在过表达NCAM1细胞爬片和pCDNA3.1细胞爬片均无阳性信号,表明回收自身抗体可特异性识别过表达细胞爬片上的NCAM1抗原;由图12可以看出,回收的患者1自身抗体的洗脱液在大鼠脑组织海马和皮层部位上的阳性信号强于回收的对照洗脱液在此部位的信号,表明回收的自身抗可特异性识别大鼠脑组织上表达的NCAM1抗原。
实施例6抗NCAM1自身抗体在疑似神经系统自身免疫疾病样本的检出率
选取226例具有头痛、眩晕、恶心、呕吐,复视,步态肢体共济失调,构音障碍,眼球震颤,记忆力障碍,智能减退,近端肌无力,四肢肌肉缺乏协调性、痴呆,癫痫等其中一种或多种症状的患者(Hu、Yo、CV2、Ma2、Amphiphysin、Ma1、SOX1、NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、D2R、Neurexin3、KCNA4、GABAARγ2、ATP1A3、Homer3、ARHGAP26、ITPR1/2、mGluR1、CARP VIII、AP3B2、septin5、GM1,GD1b,GQ1b、Sulfatides、GT1b、GT1a、GD3、GD2、GD1a、GM4、GM3、GM2自身抗体检测为阴性的患者),使用实施例3中制备好的过表达NCAM1的细胞爬片进行免疫荧光检测,具体步骤参考实施例3;
结果:免疫荧光结果显示,在226例疑似患有神经系统症状的患者中检出9例抗NCAM1自身抗体阳性患者,阳性率为3.98%,9例抗NCAM1自身抗体阳性患者的筛查结果见图13,9例患者中5例确诊为副肿瘤综合征,4例确诊为自身免疫性脑炎。
结论:抗NCAM1自身抗体可在患有神经系统症状的患者中检出,说明该自身抗体对副肿瘤综合征和自身免疫性脑炎的诊断具有辅助作用。
实施例7大鼠NCAM1检测患者血清中NCAM1自身抗体
本实施例查找与人NCAM1基因同源性较高的大鼠NCAM1基因,参考实施例3进行载体构建,制备过表达重组鼠源NCAM1的细胞爬片,使用实施例6中的9例抗NCAM1自身抗体阳性患者样本进行免疫荧光检测,9例阳性患者使用过表达重组鼠源NCAM1的细胞爬片均检出阳性信号。大鼠NCAM1蛋白序列如SEQ ID NO.3所示,大鼠NCAM1基因序列如SEQ ID NO.4所示。
结论:与人NCAM1基因同源性较高的大鼠NCAM1基因表达的蛋白依然可以被患者血清中的自身抗体所识别。
实施例8基于细胞的免疫荧光法验证抗NCAM1自身抗体的特异性
选取具有以下一种或多种神经系统自身免疫病(Hu、Yo、CV2、Ma2、Amphiphysin、Ma1、SOX1、NMDAR、AMPAR1、AMPAR2、LGI1、CASPR2、GABABR、DPPX、IgLON5、D2R、Neurexin3、KCNA4、GABAARγ2、ATP1A3、Homer3、ARHGAP26、ITPR1/2、mGluR1、CARP VIII、AP3B2、septin5、GM1,GD1b,GQ1b、Sulfatides、GT1b、GT1a、GD3、GD2、GD1a、GM4、GM3、GM2)50例患者和30例健康对照的血清,使用实施例3中制备好的过表达NCAM1的细胞爬片进行免疫荧光检测,具体步骤参考实施例3;
结果:免疫荧光结果显示,所选取的50例神经系统自身免疫病患者和30例健康对照的血清均不与过表达NCAM1细胞爬片产生类似于实施例3中患者1或患者2血清相似的细胞形态。
结论:患有本发明描述的神经系统疾病的患者具有针对NCAM1蛋白的自身抗体,而另一些患有神经系统疾病的患者和对照样本没有这种抗体。本发明为实现神经系统疾病的诊断提供了待测的与自身抗体结合的新抗原,说明该抗体对副肿瘤综合征或自身免疫性脑炎的诊断具有辅助作用。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
Claims (10)
1.一种自身免疫性疾病标志物在制备用于神经系统相关疾病诊断的检测试剂或检测试剂盒中的应用,其特征在于,所述自身免疫性疾病标志物为与NCAM1蛋白结合的自身抗体,在检测时是检测样品与NCAM1蛋白结合的自身抗体。
2.如权利要求1所述的应用,其特征在于,所述NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
3.如权利要求2所述的应用,其特征在于,编码SEQ ID NO.1所示的氨基酸序列的核苷酸序列包括Ⅰ)~Ⅲ)中的任意一种:
Ⅰ)SEQ ID NO.2所示的核苷酸序列;
Ⅱ)SEQ ID NO.2所示的核苷酸序列的10%~80%,且编码识别NCAM1自身抗体的氨基酸序列;
Ⅲ)Ⅰ)或Ⅱ)中的核苷酸序列突变后,且编码识别NCAM1自身抗体的氨基酸序列。
4.如权利要求2所述的应用,其特征在于,所述神经系统相关疾病包括副肿瘤综合征、小脑变性和边缘性脑炎。
5.如权利要求4所述的应用,其特征在于,所述神经系统相关疾病包括头痛,眩晕、恶心、呕吐,复视,步态肢体共济失调,构音障碍,眼球震颤,记忆力障碍,智能减退、近端肌无力或四肢肌肉缺乏协调性的症状。
6.如权利要求1所述的应用,其特征在于,检测样品为全血、血清和脑脊液的一种或几种。
7.一种检测自身免疫性疾病的蛋白在制备用于神经系统相关疾病诊断的检测试剂或检测试剂盒中的应用,其特征在于,所述检测自身免疫性疾病的蛋白为衍生自NCAM1蛋白的一个或多个表位,或者为融合其他氨基酸的融合蛋白;其中,NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
8.一种检测神经系统相关疾病的试剂盒,其特征在于,包括检测抗NCAM1抗体的试剂和标记抗体;其中:
所述检测抗NCAM1抗体的试剂包括NCAM1蛋白、表达NCAM1蛋白的细胞、表达NCAM1蛋白的组织和含有NCAM1蛋白的裂解物中的一种或多种;
所述标记抗体为能够与人IgG的Fc片段结合的抗体。
9.根据权利要求8所述的检测神经系统相关疾病的试剂盒,其特征在于,所述NCAM1蛋白的氨基酸序列包括a)~c)中的任意一种:
a)如SEQ ID NO.1所示的氨基酸序列;
b)如SEQ ID NO.1所示的氨基酸序列的10%~80%,且识别NCAM1自身抗体的氨基酸序列;
c)经a)或b)中的氨基酸序列经修饰或突变后,且识别NCAM1自身抗体的氨基酸序列。
10.根据权利要求8所述的检测神经系统相关疾病的试剂盒,其特征在于,所述标记抗体包括辣根过氧化物酶标记抗人抗体、碱性磷酸酶标记抗人抗体、生物素标记的抗人抗体、FITC标记的抗人抗体或Alexa Fluor染料标记的抗人抗体。
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