CN112540178B - 一种检测早期老年痴呆症的免疫组化试剂盒及其使用方法 - Google Patents
一种检测早期老年痴呆症的免疫组化试剂盒及其使用方法 Download PDFInfo
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Abstract
本发明涉及一种检测试剂盒，具体涉及一种检测早期老年痴呆症的免疫组化试剂盒及其使用方法。包括以下试剂：试剂A：APOE4点突变单克隆抗体，试剂B：HRP标记的二抗工作液，试剂C：过氧化物酶失活剂，试剂D：聚合物增强剂，PBST洗涤液，抗原修复液，DAB显色液及复染工作液；其中APOE4点突变单克隆抗体重链氨基酸序列如SEQ ID NO：3所示，轻链氨基酸序列如SEQ ID NO：4所示。本发明所提供的免疫组化试剂盒，所用的抗体为筛选得到的特异性强、灵敏度高的APOE4点突变单克隆抗体，定性定位准确，背景清晰，操作方便，孵育时间短，适宜进行扩大化生产并用于临床诊断早期老年痴呆症。
Description
技术领域
本发明涉及一种检测试剂盒，具体涉及一种检测早期老年痴呆症的免疫组化试剂盒及其使用方法。
背景技术
阿尔茨海默病(Alzheimer's disease,AD)，又名老年痴呆症，是一种年龄相关的神经退行性疾病，其主要临床表现为记忆功能障碍和行为方面的改变。随着人口老龄化的加剧，中国AD患者数目急剧增加，到2015年已达950万，成为全球痴呆症患者人数最多的地区，给社会和家庭带来巨大的负担。
作为一种进行性的疾病，AD最重要的生理特征是大脑部分区域经历进行性功能障碍、神经元损失和突触的丧失。Bateman等的研究证明，在AD发作前10至20年患者大脑就开始逐渐产生变化。而这些功能性的变化往往难以逆转，这也导致了目前AD治疗的困境。因此，对早期AD的检测可能是预防与治疗AD最有效的手段。
血液生物标记物的检测因其便宜、易于操作以及非侵入性的优势成为众多疾病检测的首选方式。而在AD中，众多蛋白被证明在AD患者血浆中有明显变化，如BDNF,AGT,IGFBP-2,OPN等，提示血浆蛋白质含量检测可能是AD早期诊断有价值的方式。
而载脂蛋白E(ApoE)被证明与AD发生相关。载脂蛋白(Apo)E基因分为三种等位基因型ε2、ε3、ε4，分别编码ApoE2、ApoE3和ApoE4三种异构体。三种不同亚型ApoE之间的区别仅在于其一级结构112与158位点的氨基酸不同，即ApoE2(112Cys,158Cys)、ApoE3(112Cys,158Arg)、ApoE4(112Arg,158Arg)。其中，ApoEε4基因高表达已被公认为是AD最大的危险因素之一。1990年Duke大学对占发病大部分的散发性AD(SAD)和迟发家族性AD(FAD)做免疫组化分析发现:老年斑和神经纤维缠结中存在ApoE4。研究证明,散发性和家族性晚发AD均与ε4有关。临床发现杂合子ε4携带者患晚发AD的机率比非携带者高3倍、纯合子ε4携带者患AD的机率比非携带者高10-20倍。carwfodr等发现ε4基因与患者年龄、发病年龄有相关性。而在早期诊断中，若个体携带ApoE4,则给AD的诊断增加至少5％～10％的置信度,尤其对于不同痴呆类型的鉴别诊断。同时若患者具备AD相关行为表型，且携带ApoE4,则诊断AD的准确性可达94％～98％。这些均提示：ApoE4基因型的检测可能是早期AD诊断最有价值的指标之一。
但是，目前国内外对ApoE基因型的检测主要通过第二代高通量测序系统，成本高昂的同时，准确性亦难得到保证。据2015年底全国性肿瘤二代测序实验室质量评估报告结果显示，55％的实验室测序结果不合格，22％的实验室质量评估为0分。这些导致ApoE4基因型检测在AD诊断中难以被推广使用。
而基因型的变化会导致蛋白表达的变化。据文献报道,ApoE等位基因的差异可引起AD患者血清抑ApoE浓度发生变化,并使AD患者血脂代谢发生异常；AD患者血清ApoE4浓度较正常人显著升高。提示检测血清中ApoE4蛋白的含量可能在早期AD诊断中具有非常重要的意义。而目前国内外并未有ApoE4蛋白检测试剂盒面市。与此同时，蛋白质试剂盒检测具备准确度高、成本低廉的优势。因此，研究者认为血清ApoE4蛋白检测试剂盒的研发能为早期AD诊断提供有力的证据，具备一定的现实意义及经济效益。单克隆抗体技术在疾病诊断、药物开发中的应用是目前医药领域的一个研究热点，也为多种疾病的治疗提供了新的途径。
综上所述，开发一种能够用于临床诊断的检测早期老年痴呆症的免疫组化试剂盒具有重要意义。
发明内容
本发明针对现有技术中存在的技术问题，提供一种能够用于临床诊断的检测早期老年痴呆症的免疫组化试剂盒。
本发明解决上述技术问题的技术方案如下：
本发明所提供的免疫组化试剂盒，包括以下试剂：
试剂A：APOE4点突变单克隆抗体，
试剂B：HRP标记的二抗工作液，
试剂C：过氧化物酶失活剂，
试剂D：聚合物增强剂，
PBST洗涤液，抗原修复液，DAB显色液及复染工作液；
其中APOE4点突变单克隆抗体重链氨基酸序列如SEQ ID NO：3所示，轻链氨基酸序列如SEQ ID NO：4所示。
其中，所述APOE4点突变单克隆抗体由保藏于中国典型培养物保藏中心CCTCC，保藏编号为：CCTCC NO：C2020121的杂交瘤细胞株ACE8产生。
在本发明的一个实施例中，试剂A为3％BSA-PBST配制的APOE4单克隆抗体液体，浓度为0.2μg/μL。
在本发明的一个实施例中，试剂B为HRP标记的抗鼠IgG聚合物，浓度为1mg/ml。
在本发明的一个实施例中，试剂C过氧化物酶失活剂为3％H2O2-甲醇溶液。
在本发明的一个实施例中，试剂D中的聚合物增强剂为PEG4000。
在本发明的一个实施例中，所述PBST洗涤液为含有体积百分比为0.01％Tween-20的0.01mol/L PBS缓冲液。
在本发明的一个实施例中，所述抗原修复液为10mmol/L的枸橼酸盐缓冲液，pH6.0。
本发明还提供了上述检测早期老年痴呆症的免疫组化试剂盒的使用方法，包括以下步骤：
(1)组织切片脱蜡和水化：二甲苯5min×2次→无水乙醇5min×1次→95％乙醇5min×1次→80％乙醇5min→70％乙醇5min→自来水冲洗片刻→PBST洗涤液冲洗1min×3次；
(2)抗原修复：压力锅中加入足以淹没切片的抗原修复液，加热至沸腾，放入切片，扣上压力阀，继续加热至喷气，维持1.5min，取出自然冷却至室温；
(3)洗涤：ddH2O浸泡5分钟，清洗2次，PBST洗涤液浸泡5分钟，清洗2次；
(4)灭活酶：每片滴加试剂C酶失活剂工作液，室温处理15min；
(5)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(6)加一抗：加入试剂A，37℃，湿盒中孵育1小时；
(7)洗涤；PBST洗涤液浸泡5分钟，清洗3次；
(8)加聚合物增强剂：加入试剂D聚合物增强剂50μl，湿盒中室温孵育30分钟；
(9)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(10)加酶标二抗：加入试剂B HRP标记的抗鼠IgG聚合物二抗工作液，室温37℃，孵育30分钟；
(11)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(12)显色：每张切片滴加100μLDAB显色液，同时在显微镜下观察控制显色程度，待出现阳性反应，则置流水下冲洗及时终止反应；
(13)复染：将切片放入复染工作液中，染色30min,用蒸馏水冲洗干净；1％盐酸-乙醇分化，流水冲洗；脱水、封片。
本发明所提供的免疫组化试剂盒，所用的抗体为筛选得到的特异性强、灵敏度高的APOE4点突变单克隆抗体，定性定位准确，背景清晰，操作方便，孵育时间短，适宜进行扩大化生产并用于临床诊断早期老年痴呆症。
附图说明
图1为纯化后的抗体SDS-PAEG电泳图谱，其中，MW：表示分子量；泳道1表示ACE8未经煮沸变性；泳道2；表示ACE8经煮沸变性；
图2为Western blot分析转染的HeLa细胞表达全长人类ApoE2(hApoE2),ApoE3(hApoE3)或ApoE4(hApoE4)的提取物，其中上图中采用的一抗为本发明的杂交瘤细胞ACE8所产生的单抗，下图采用的一抗为panApoE抗体；
图3为使用ApoE4(ACE8)mouse mAb鼠单抗对原代大鼠海马神经细胞进行免疫荧光分析结果图；
图4为使用ApoE4(ACE8)mouse mAb鼠单抗对人脑胶质瘤U87细胞免疫荧光细胞染色；
图5为使用ApoE4(ACE8)mouse mAb鼠单抗对石蜡包埋的人皮肤进行免疫组化分析结果图，其中A为对照肽，B为抗原特异性肽；
图6为使用ApoE4(ACE8)mouse mAb鼠单抗对石蜡包埋的人肾组织切片免疫组化分析结果图；
图7为使用ApoE4(ACE8)mouse mAb鼠单抗对石蜡包埋的人脾组织切片免疫组化分析结果图；
图8为使用ApoE4(ACE8)mouse mAb鼠单抗对石蜡包埋的人结肠组织切片免疫组化分析结果图。
具体实施方式
以下结合具体实施方式对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1免疫复合物制备
(1)通过固相法合成APOE4蛋白的16肽(APOE4蛋白第109-124位氨基酸序列：EDVRGRLVQYRGEVQA，如SEQ ID NO：5所示)，在美国ABI公司的多肽合成仪(431A)上进行，采用Fmoc(9-芴甲氧羰基)方案，合成步骤按照ABI公司的多肽合成操作手册进行。经高效液相色谱纯化，序列鉴定通过质谱分析制备得到APOE4蛋白的表位肽。
(2)通过偶联试剂Sulfo-SMCC将制备得到的ApoE4蛋白的表位肽和KLH偶联，得到免疫复合物。
以上采用本领域常见试剂、仪器等即可实现。
实施例2单克隆抗体制备
(1)动物免疫
使用6～8周龄雌性Balb/c纯系小鼠六只，按照如下免疫方法进行五次免疫注射。
第一次免疫：第0天，用2ml注射器向每只小鼠腹股沟皮下注射0.2ml乳化液，其中乳化液为免疫复合物溶液和弗氏完全佐剂按1：1混合制备得到，乳化液中含30～50μg免疫复合物，免疫复合物溶液用1×PBS稀释免疫复合物得到。
第二次免疫：第21天，用2ml注射器向每只小鼠腹股沟皮下注射0.2ml乳化液，其中乳化液为免疫复合物溶液和弗氏不完全佐剂按1：1混合制备得到，乳化液中含30～50μg免疫复合物，免疫复合物溶液用1×PBS稀释免疫复合物得到。
第三次免疫：第42天，用2ml注射器向每只小鼠腹股沟皮下注射0.2ml乳化液，其中乳化液为免疫复合物溶液和弗氏不完全佐剂按1：1混合制备得到，乳化液中含30～50μg免疫复合物，免疫复合物溶液用1×PBS稀释免疫复合物得到。
第四次免疫：第63天，用2ml注射器向每只小鼠腹股沟皮下注射0.2ml乳化液，其中乳化液为免疫复合物溶液和弗氏不完全佐剂按1：1混合制备得到，乳化液中含30～50μg免疫复合物，免疫复合物溶液用1×PBS稀释免疫复合物得到。
第五次免疫：第78天，用2ml注射器向每只小鼠尾静脉注射0.2ml乳化液，其中乳化液为免疫复合物溶液和弗氏不完全佐剂按1：1混合制备得到，乳化液中含30～50μg免疫复合物，免疫复合物溶液用1×PBS稀释免疫复合物得到。
分别在第1天、第52天、第73天采用间接ELISA法测试免疫血清效价，其中第73天的效价达到了128K以上，在第78天对小鼠尾静脉进行冲击免疫，并于3天后(第81天)取小鼠脾脏B细胞进行融合。
(2)杂交瘤细胞的制备
小鼠骨髓瘤细胞SP2/0的制备：把来自BALB/C小鼠的SP2/0骨髓瘤细胞株以含10％FBS-DMEM培养基培养传代，在含5％CO2饱和湿度的37℃培养箱中培养。融合前一天传代以保证融合时细胞进入对数生长期。
饲养细胞的制备：采取小鼠腹腔的巨噬细胞作为饲养层细胞。取正常的BALB/c小鼠，摘除眼球采血，并分离血清作为抗体检测时的阴性对照血清。同时通过颈脱位致死小鼠，浸泡于75％酒精中5分钟，于解剖台板上固定后掀开左侧腹部皮肤，可看到脾脏，换眼科剪镊，在超净台中用无菌手术剪剪开腹膜，取出脾脏置于平皿中，并细心剥去周围结缔组织。用注射器内芯挤压脾脏，使细胞分散，用10ml不完全培养基吹洗细胞数次，制成单细胞悬液。转移入50ml离心管，1000r/min离心5-10分钟，用不完全培养基离心洗涤1-2次，然后将细胞重悬于10ml不完全培养基混匀，取上述悬液，加台酚蓝染液作活细胞计数后备用。通常每只小鼠可得1×108-2.5×108个脾细胞，将上述细胞悬液加入96孔板，每孔0.1ml(相当于2滴)，然后置37℃4％CO2的培养箱中培养。
免疫脾细胞的制备：将经过免疫的Balb/c小鼠拉颈或CO2处死，于75％乙醇溶液中浸泡5分钟，超净台内无菌开腹取出脾脏，以5ml RPMI1640不完全培养液清洗一次；在盛有20ml不完全培养液的平皿中放置不锈钢网，并将脾脏移入到筛网上，用注射器内芯轻轻研磨脾脏，吸取平皿内培养基液轻轻冲洗不锈钢筛网，使脾细胞全部通过网孔进入到溶液中；将上述脾细胞溶液转移至50ml离心管中，加不完全培养液15-20ml，混匀；1200r/min离心10分钟，弃上清；细胞沉淀再用不完全培养液同法离心洗涤一次后重悬，锥虫蓝染色做活细胞计数。一般免疫脾细胞体积约为正常脾脏体积的2倍，每只小鼠可得1.0×108-2.5×108个脾细胞。
细胞融合：取对数生长期的骨髓瘤细胞SP2/0，1000r/min离心5分钟，弃上清，用不完全培养液重悬细胞沉淀，混匀后锥虫蓝染色作或细胞计数，取所需细胞，用不完全培养液洗涤2次；取制备好的免疫脾细胞用不完全培养液洗涤2次；将骨髓瘤细胞与脾细胞按1:10或1:5的比例加入同一个50ml离心管内，补加不完全培养液至30-40ml，充分混匀；1200r/min离心10分钟，弃上清，轻轻弹击离心管底，使细胞沉淀松散呈均匀糊状。室温下融合：一手均匀地转动离心管，另一手用1ml吸管吸取37℃预热的45％PEG60001ml，并沿转动的离心管管壁均匀、缓慢地加入，时间控制在60秒左右，然后立即轻柔地将细胞悬液全部吸入吸管，时间控制在30秒，静置30秒，再将其轻柔地吹入离心管内，时间控制30秒左右，立即在五分钟内按每分钟1、2、4、8和10ml的体积(共25ml)加入预热至37℃的不完全培养液，通过稀释PEG而终止其促细胞融合作用。800r/min离心6分钟，弃上清，加入适量20％FCS-RPMI1640完全培养液轻轻混悬融合细胞，切勿用力吹吸细胞。按10ml一块96孔细胞培养板计算完全培养液用量。
将融合后的细胞悬液加入已铺有饲养细胞的96孔细胞培养板中，100μl/孔，37℃，5％CO2培养箱培养。通常1只免疫鼠脾细胞融合后可加4-6块96孔板。
(3)杂交瘤细胞的筛选与克隆
每隔4天换1/2培养液(HAT)一次，10天后改用含HT的选择性培养液。融合后的杂交瘤细胞在含HT的选择性培养液中大约持续培养两周。在细胞集落长到适当大小时(在10倍物镜下观察，细胞克隆大小以占满一个视野为宜)，吸取培养液上清，适当稀释后做间接ELISA，筛选阳性克隆。
a、初步筛选
包被表位多肽APOE4-112Arg(APOE4蛋白第109-124位氨基酸序列：EDVRGRLVQYRGEVQA，作为正筛选)和APOE4-112Cys(EDVCGRLVQYRGEVQA作为负筛选)，作为负筛选的表位多肽同样采用固相法合成，采用间接ELISA方法进行筛选，对APOE4-112Arg多肽有阳性反应，而对APOE4-112Cys无反应的细胞株为初筛的阳性细胞株，阳性细胞株可按照b步骤作进一步的筛选。
b、二次筛选
将含有编码APOE4-112Cys，也即ApoE2(112Cys,158Cys)蛋白、ApoE3(112Cys,158Arg)蛋白和编码APOE4-112Arg，也即ApoE4(112Arg,158Arg)蛋白的核苷酸序列分别(其中编码ApoE2蛋白的核苷酸序列如SEQ ID NO：6所示，编码的ApoE2蛋白的氨基酸序列如SEQID NO：9所示；编码ApoE3蛋白的核苷酸序列如SEQ ID NO：7所示，编码的ApoE2蛋白的氨基酸序列如SEQ ID NO：10所示；编码ApoE4蛋白的核苷酸序列如SEQ ID NO：8所示，编码的ApoE2蛋白的氨基酸序列如SEQ ID NO：11所示)克隆到pcDNA3.1(+)全长表达质粒上并按照标准分子生物学方法制备质粒，将质粒用瞬时转染方法感染HEK293细胞(购自ATCC，美国)，然后裂解细胞，提纯所表达的蛋白。
包被上述三种蛋白，采用间接ELISA方法进一步的筛选初筛的阳性细胞株，对APOE4-112Arg蛋白有阳性反应，而对APOE4-112Cys蛋白无反应的细胞株视为阳性细胞株。
对筛选出来的阳性细胞株通过有限稀释法进行克隆化。挑选单集落的克隆利用a和b的方法进一步确定阳性的细胞株。
制备杂交瘤细胞悬液：轻轻吹散ELISA检测为阳性孔中的杂交瘤细胞，将杂交瘤细胞用移液器吸入到10mL的离心管中，计数，计算，将细胞浓度以完全培养基稀释到200个/mL，取出2mL加入到2mL含有饲养细胞的完全培养基中，杂交瘤细胞的浓度则变为100个/mL；
有限稀释：将上述杂交瘤细胞悬液以每孔100μL加入到96孔细胞培养板的第1、2两行，这两行的杂交瘤细胞的浓度为每孔10个。在剩余细胞悬液中加入等体积的饲养细胞悬液，混匀后加入到第3、4两行，则这两行的杂交瘤细胞的浓度为每孔5个，重复上述步骤，则第5、6两行的浓度为每孔2.5个，第7、8两行的浓度为每孔1.25个。培养3天后，在显微镜下观察细胞生长状况，并小心吸取培养板上100μL的培养液，再添加完全培养基100μL。利用间接ELISA对孔中上清进行检测，选择效价高的细胞培养孔进行再次克隆化，并尽可能选取单克隆孔。直到连续两次克隆化的单克隆孔检测全为阳性为止。
细胞融合获得6株能稳定分泌抗APOE4点突变单克隆抗体的杂交瘤细胞株，见下表1。其中，杂交瘤细胞株ACE8所分泌的抗体在用于检测APOE4蛋白时效果最优，将其于2020年7月22日保藏在中国典型培养物保藏中心，保藏编号为CCTCCNO:C2020121。
表1筛选得到的稳定分泌抗ApoE4蛋白单克隆抗体的杂交瘤细胞株
| 杂交瘤细胞株 | 杂交瘤细胞株 | 杂交瘤细胞株 |
| ACE8 | ACE4 | ACE14 |
| ACE23 | ACE36 | ACE72 |
按照间接ELISA方法对杂交瘤细胞株上清效价进行测定，从第3孔至第10孔以0.01M pH7.2的PBS缓冲液2倍逐级稀释。第1孔以融合时小鼠血清稀释至100倍作阳性对照，第2孔以RPMI 1640完全培养液作阴性对照，阴性对照OD值＜0.2，阳性对照OD值＞1.8为检测系统有效，当OD值≥2×阴性对照OD值时，为阳性，反之为阴性。检测值最低阳性孔所对应的稀释比例即为杂交瘤细胞株培养上清效价，此杂交瘤细胞株培养上清效价可达4096K(K,表示1000,4096K表示4096000)，见下表2。
表2杂交瘤细胞株上清效价结果
(4)腹水生产和单克隆抗体的纯化
腹水的诱生：在接种杂交瘤细胞前1周，先用0.3-0.5ml/只液体石蜡接种至小鼠腹腔，使小鼠致敏。7-10天后用PBS或无血清培养基稀释的杂交瘤细胞接种至小鼠腹腔，每只小鼠3×105/0.5ml。接种后每天观察小鼠腹水产生情况，如腹部明显膨大，以手触摸时，皮肤有紧张感，即可拉颈处死小鼠，用滴管将腹水吸入15ml离心管中，一只小鼠可获1-5ml腹水。收集的腹水离心取上清，取小样放于-20℃以下冰箱保存备用。
单克隆抗体的纯化：将收集的腹水按照硫酸铵沉淀方法纯化腹水。SDS-PAGE对抗体进行纯度验证和定量，结果见附图1。
具体纯化步骤为：1)1份腹水加入2份PBS进行稀释；2)然后根据稀释后的总体积，按照0.277g硫酸铵/ml加入到腹水稀释液中(这个加入硫酸铵的过程需要在冰水浴中进行，分多次加入，边加边摇溶，在30分钟左右加完)；3)然后静置于4度两个小时或者过夜；4)离心12000rpm，10min或者3000rpm，20min，弃上清，用PBS复溶至原体积；5)重复步骤2，3；6)离心12000rpm，10min或者3000rpm，20min，弃上清，用适量PBS溶解，测定浓度，SDS-PAGE检测纯度，检测完毕后加入40-50％的甘油。
(5)腹水效价测定
按照间接ELISA方法对腹水效价进行测定，从第3孔至第10孔以0.01M pH7.2的PBS缓冲液2倍逐级稀释。第1孔以融合时小鼠血清稀释至100倍作阳性对照，第2孔以RPMI 1640完全培养液作阴性对照，阴性对照OD值＜0.2，阳性对照OD值＞1.8为检测系统有效，当OD值≥2×阴性对照OD值时，为阳性，反之为阴性。检测值最低阳性孔所对应的稀释比例即为腹水效价，腹水效价可达4096K(K,表示1000,4096K表示4096000)。
(6)抗体效价测定
按照间接ELISA方法对抗体效价进行测定，从第3孔至第10孔以0.01M pH7.2的PBS缓冲液2倍逐级稀释。第1孔以融合时小鼠血清稀释至100倍作阳性对照，第2孔以RPMI 1640完全培养液作阴性对照，阴性对照OD值＜0.2，阳性对照OD值＞1.8为检测系统有效，当OD值≥2×阴性对照OD值时，为阳性，反之为阴性。检测值最低阳性孔所对应的稀释比例即为抗体效价，此抗体效价可达4096K(K,表示1000,4096K表示4096000)。(7)单克隆抗体的亚型鉴定:采用Serotec公司的小鼠单克隆抗体免疫球蛋白分型试剂盒分析。将纯化的单抗作适当稀释后进行检测，操作严格按试剂盒说明书进行。试验结果如下表3所示(数据源A：吸光度450nm)：
表3亚型检测结果
| 板孔 | 吸光度 | 亚型 |
| A | 2.31 | Ig1 |
| B | 0.07 | Ig2a |
| C | 0.08 | Ig2b |
| D | 0.03 | Ig2c |
| E | 0.06 | Ig3 |
| F | 0.06 | IgA |
| G | 0.04 | IgM |
| H | 0.01 | PBS |
杂交瘤细胞株ACE8分泌的单克隆抗体为γ1型，与APOE4的抗原肽(第109-124位氨基酸序列)具有高的特异性和灵敏性。杂交瘤细胞株ACE8已由中国典型培养物保藏中心(CCTCC)保藏；地址：中国武汉武汉大学；保藏日期：2020年7月22日；保藏编号为：CCTCCNO：C2020121。
实施例3单克隆抗体的鉴定
(1)免疫印迹鉴定
分别构建包含APOE4-112Cys，也即ApoE2(112Cys,158Cys)蛋白、ApoE3(112Cys,158Arg)蛋白和APOE4-112Arg，也即ApoE4(112Arg,158Arg)蛋白的核苷酸序列的pcDNA3.1(+)全长表达质粒，将质粒分别转染HEK-293细胞，培养细胞裂解液用该单抗进行WesternBlotting检测，在相应位置呈现目的条带，表明检测到ApoE4-112Arg蛋白的表达。具体步骤如下：
a、SDS聚丙烯酰胺凝胶电泳：方法参见F.奥斯伯等《精编分子生物学实验指南》(科学出版社1998)。采用10％分离胶和5％浓缩胶，电泳条件为电压150V，溴酚蓝染料带距离胶底边1.5厘米左右终止电泳。
b、电转移：方法参见F.奥斯伯等《精编分子生物学实验指南》(科学出版社1998)。用电转移方式将其转移至PVDF(0.45μm)膜上。
c、免疫印迹：电转膜结束后，膜在5％脱脂奶粉封闭液中室温封闭1小时后，用本发明制备的单抗(1μg/ml)作为一抗，室温孵育2小时或4℃反应过夜，用TBST(TBS加入0.5％Tween-20)洗涤3次，每次10min。以辣根过氧化物酶标记的羊抗鼠IgG作为二抗，室温反应2h，用上述方法洗涤后用ECL作用1min，于全自动化学发光成像仪(Bio-RadVersaDoc5000MP)曝光成像。
结果见图2，图2为Westernblot分析转染的HeLa细胞提取物用表达全长人类ApoE2(hApoE2),ApoE3(hApoE3)或ApoE4(hApoE4)，其中上图中采用的一抗为本发明的杂交瘤细胞ACE8所产生的单抗，下图采用的一抗为pan ApoE抗体(购自武汉艾美捷生物科技有限公司)。ApoE2(112Cys,158Cys)、ApoE3(112Cys,158Arg)为对照，均未与抗体结合，ApoE4(112Arg,158Arg)特异性的与抗体结合。
(2)免疫荧光细胞染色鉴定
分别构建包含APOE4-112Cys，也即ApoE2(112Cys,158Cys)蛋白、ApoE3(112Cys,158Arg)蛋白和APOE4-112Arg，也即ApoE4(112Arg,158Arg)蛋白的核苷酸序列的pcDNA3.1(+)全长表达质粒，将质粒分别转染原代大鼠海马神经细胞/人脑胶质瘤Μ87细胞，按照免疫荧光技术流程验证APOE4-112Arg阳性的抗体，结果见图3、4所示。
(3)杂交瘤细胞株ACE8、抗APOE4点突变单克隆抗体序列测定
提取杂交瘤细胞株ACE8的总RNA，用通用引物反转录成cDNA，再扩增抗体的轻链和重链，把轻链和重链分离出来，克隆到标准克隆载体上表达，单菌落PCR鉴定轻链和重链，挑选5个轻链和重链长度正确的单菌落进行测序，5次测序结果几乎相同，则认为是抗体的真实序列。(测序过程和抗体大量表达委托南京金斯瑞生物科技公司完成)
杂交瘤细胞株ACE8得到的基因序列结果：抗APOE4点突变单克隆抗体重链编码基因序列长1332bp，序列如SEQ ID NO:1所示；抗APOE4点突变单克隆抗体轻链编码基因序列长657bp，序列如SEQ ID NO:2所示。根据所获得的基因序列推导出该基因序列所编码的重链由444个氨基酸组成，序列如SEQ ID NO:3所示；轻链由219个氨基酸组成，序列如SEQ IDNO:4所示。
实施例4免疫组化试剂盒
一种检测早期老年痴呆症的免疫组化试剂盒，包括以下试剂：
试剂A：APOE4点突变单克隆抗体，具体为3％BSA-PBST配制的APOE4单克隆抗体液体，浓度为0.2μg/μL；
试剂B：HRP标记的二抗工作液，具体为HRP标记的抗鼠IgG聚合物，浓度为1mg/ml；
试剂C：过氧化物酶失活剂，具体为3％H2O2-甲醇溶液；
试剂D：聚合物增强剂，具体为PEG4000；
PBST洗涤液，具体为含有体积百分比为0.01％Tween-20的0.01mol/L PBS缓冲液；
抗原修复液，具体为10mmol/L的枸橼酸盐缓冲液，pH6.0；
DAB显色液；及复染工作液，具体为苏木素复染工作液；
其中APOE4点突变单克隆抗体为实施例2中制备得到的单克隆抗体。
上述检测早期老年痴呆症的免疫组化试剂盒的使用方法，包括以下步骤：
(1)组织切片脱蜡和水化：二甲苯5min×2次→无水乙醇5min×1次→95％乙醇5min×1次→80％乙醇5min→70％乙醇5min→自来水冲洗片刻→PBST洗涤液冲洗1min×3次；
(2)抗原修复：压力锅中加入足以淹没切片的抗原修复液，加热至沸腾，放入切片，扣上压力阀，继续加热至喷气，维持1.5min，取出自然冷却至室温；
(3)洗涤：ddH2O浸泡5分钟，清洗2次，PBST洗涤液浸泡5分钟，清洗2次；
(4)灭活酶：每片滴加试剂C酶失活剂工作液，室温处理15min；
(5)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(6)加一抗：加入试剂A，37℃，湿盒中孵育1小时；
(7)洗涤；PBST洗涤液浸泡5分钟，清洗3次；
(8)加聚合物增强剂：加入试剂D聚合物增强剂50μl，湿盒中室温孵育30分钟；
(9)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(10)加酶标二抗：加入试剂B HRP标记的抗鼠IgG聚合物二抗工作液，室温37℃，孵育30分钟；
(11)洗涤：PBST洗涤液浸泡5分钟，清洗3次；
(12)显色：每张切片滴加100μl DAB显色液，同时在显微镜下观察控制显色程度，待出现阳性反应，则置流水下冲洗及时终止反应；
(13)复染：将切片放入复染工作液中，染色30min,用蒸馏水冲洗干净；1％盐酸-乙醇分化，流水冲洗；脱水、封片。
取部分老年痴呆症患者的人皮肤，人肾，人脾，结肠切片(均从武汉大学中南医院获得)，将切片按照如下步骤进行处理后置于显微镜下观察：结果见图5、6、7、8，其中图5中A为对照肽，其氨基酸序列为VERDVGRLRVQQGYEA，B为抗原特异性肽，其氨基酸序列为EDVRGRLVQYRGEVQA，上述对照肽和抗原特异性肽均采用固相法合成。
间接ELISA检测方法操作程序：
1)取96孔酶标板，每孔加入用包被液稀释的包被浓度的免疫复合物50μl，即100ng的抗原，边沿孔应尽量避免使用，会降低吸光值影响结果，4℃过夜或者37℃孵育2小时；
2)倒去抗原，每孔加入100μl封闭液(1％BSA，0.1MKpi,0.1％Tween-20,0.02％柳硫汞，pH 7)，4℃过夜或者37℃孵育2小时，倒去封闭液，洗涤液(0.1M KPi,0.05％Tween-20,pH7)洗板3次，拍干；
3)每孔加入50μl的一抗，如果是杂交瘤细胞，则一抗就是细胞上清，如果是免疫动物的血清，则需要进行一系列的稀释(封闭液稀释)，通常起始稀释度为1:499，此后2倍的依次稀释下去，同时设置阴性对照、空白对照和阳性对照各2孔，37℃孵育1小时；洗板3次，拍干；
4)每孔加入50μl的1：1999稀释(封闭液稀释)的HRP标记的二抗，37℃孵育45min；洗板3次，拍干；
5)每孔加入100μl的TMB底物(必须现配)，室温孵育5-20分钟。
6)加入每孔100μl的终止液(0.5M草酸)，在酶标仪上读取450nm的吸光度。
免疫荧光技术流程：
1、操作前，超净工作台以紫外线照射20分钟后，开风机5分钟后方可进行后续操作；
2、将HEK293细胞用细胞消化液消化，轻轻吹打制备单个细胞悬液，细胞浓度为4×105个/ml，接种无菌96孔细胞培养板、100μl/孔；
3、将构建包含APOE4突变位点为APOE4-112Arg和APOE4-112Cys蛋白pcDNA全长表达质粒，将质粒转染HEK293细胞，同时设正常细胞空白对照(加100μl细胞生长液)；和阴性对照6孔。
4、置37℃CO2孵箱培养；逐日观察细胞并记录结果，观察5-7天。
5、将转染质粒的HEK293细胞的96孔板内液体弃去(先吸阴性孔，后吸阳性孔)，用PH7.4的PBS洗板2次，每孔150μl，洗板完毕后将96孔板放吸水纸上拍打，使孔内没有残留的液体。
6、洗板完毕后将96孔板内加入80％的冷丙酮，每孔150μl，放置-20℃固定12分钟。
7、固定完毕后将96孔板内固定液吸弃后，用PH7.4的PBS洗板一次，每孔150μl。洗板完毕后将96孔板放吸水纸上拍打，使孔内没有残留的液体。
8、洗板完毕，也可将96孔板放-20℃长期保存。
9、将96孔板内加入3％BSA，每孔150μl，放37℃30分钟，轻轻拍干孔内液体。
10、将APOE4点突变单抗用1％BSA稀释，稀释比例为1：1000倍。
11、96孔板内加入APOE4点突变单抗，每孔50μl，放37℃培养箱1小时
12、用PBST洗板3次，每孔150ml，每次间隔3-5分钟。
13、FITC标记的羊抗鼠IgG二抗的稀释，稀释比例为1：100倍(二抗注意要避光)
14、将96孔板内加入稀释好的二抗，每孔50μl，放37℃1小时。
15、用PBST洗板4次，每孔150μl，每次间隔3-5分钟，最后用纯水洗板1次，每孔150μl，洗板完毕后，将96孔板放吸水纸上拍干。
16、将96孔板内加入80％甘油封闭，每孔50μl。
17、观察结果。
所用试剂如下：
FITC标记的羊抗鼠IgG二抗，美国KPL公司产品,进口BSA。
丙酮，甘油，吐温-80，KH2PO4，NaCl，KCl，Na2HPO4·12H2O，均为国产分析纯。
洗涤液PBST的配制(pH7.4，0.05％Tween-20的PBS)；
细胞固定液的配制：丙酮80mL，超纯水20ml混匀，200ml试剂瓶中-20℃保存。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 武汉天德生物科技有限公司
<120> 一种检测早期老年痴呆症的免疫组化试剂盒及其使用方法
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1332
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caggtgcagc tggtgcagag cggcgccgag gtgaagcgcc ccggcctgag catccgcatc 60
agctgccgcc tgagcggcta caccttcacc aactaccaga tgcactgggt gcgccaggcc 120
cccggcaacc gcctggagtg gatgggcacc atctaccccg gcaacgacga caccagctac 180
aacaaccgct tccgcgagaa gatcaccgtg accctggaga ccagcctgag caccctgtac 240
atggacgcca gcagcgccaa gagcgacgac accgccgtgt actactgcgc caagggcggc 300
taccgcgcca tggagtactt cggcaacggc accgccatca ccatcagcag cgccagcacc 360
aagggcccca gcgtgttccc cctggccccc tgcagccgca gcaccagcga gagcaccgcc 420
gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480
ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540
agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcaccaagac ctacacctgc 600
aacgtggacc acaagcccag caacaccaag gtggacaagc gcgtggagag caagtacggc 660
cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 720
ccccccaagc ccaaggacac cctgatgatc agccgcaccc ccgaggtgac ctgcgtggtg 780
gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 840
gtgcacaacg ccaagaccaa gccccgcgag gagcagttca acagcaccta ccgcgtggtg 900
agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960
agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 1020
cgcgagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 1080
agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140
aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1200
ttcttcctgt acagccgcct gaccgtggac aagagccgct ggcaggaggg caacgtgttc 1260
agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1320
agcctgggca ag 1332
<210> 2
<211> 657
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaggtgatca tgaccaacag ccccgccagc gcccccatca cccccggcga gcccctgagc 60
atcagctgca agagcagcaa cagcgtgatc tacagcaacg gcaacaccta cctgggctgg 120
tacctgcaga agcccggcaa cagccccaac gccgccgtgt accgcgtgag caaccgcttc 180
agcggcgtgc ccgaccgctt cagcggcagc ggcagcggca ccgacttcac cctgaaggtg 240
agccgcgtgg aggccgagga cgtgggcatc tactactgct ggaacggcag ccacatcccc 300
tacacctggg gccagggcac caaggccgag atcaagcgca ccgtggccgc ccccagcgtg 360
ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420
ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480
agcggcaaca gccaggagag cgtgaccgag caggacagca aggacagcac ctacagcctg 540
agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600
gtgacccacc agggcctgag cagccccgtg accaagagct tcaaccgcgg cgagtgc 657
<210> 3
<211> 444
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gly Val Gly Leu Val Gly Ser Gly Ala Gly Val Leu Ala Pro Gly Leu
1 5 10 15
Ser Ile Ala Ile Ser Cys Ala Leu Ser Gly Thr Thr Pro Thr Ala Thr
20 25 30
Gly Met His Thr Val Ala Gly Ala Pro Gly Ala Ala Leu Gly Thr Met
35 40 45
Gly Thr Ile Thr Pro Gly Ala Ala Ala Thr Ser Thr Ala Ala Ala Pro
50 55 60
Ala Gly Leu Ile Thr Val Thr Leu Gly Thr Ser Leu Ser Thr Leu Thr
65 70 75 80
Met Ala Ala Ser Ser Ala Leu Ser Ala Ala Thr Ala Val Thr Thr Cys
85 90 95
Ala Leu Gly Gly Thr Ala Ala Met Gly Thr Pro Gly Ala Gly Thr Ala
100 105 110
Ile Thr Ile Ser Ser Ala Ser Thr Leu Gly Pro Ser Val Pro Pro Leu
115 120 125
Ala Pro Cys Ser Ala Ser Thr Ser Gly Ser Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Leu Ala Thr Pro Pro Gly Pro Val Thr Val Ser Thr Ala Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Pro Pro Ala Val Leu Gly Ser
165 170 175
Ser Gly Leu Thr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Leu Thr Thr Thr Cys Ala Val Ala His Leu Pro Ser Ala
195 200 205
Thr Leu Val Ala Leu Ala Val Gly Ser Leu Thr Gly Pro Pro Cys Pro
210 215 220
Pro Cys Pro Ala Pro Gly Pro Leu Gly Gly Pro Ser Val Pro Leu Pro
225 230 235 240
Pro Pro Leu Pro Leu Ala Thr Leu Met Ile Ser Ala Thr Pro Gly Val
245 250 255
Thr Cys Val Val Val Ala Val Ser Gly Gly Ala Pro Gly Val Gly Pro
260 265 270
Ala Thr Thr Val Ala Gly Val Gly Val His Ala Ala Leu Thr Leu Pro
275 280 285
Ala Gly Gly Gly Pro Ala Ser Thr Thr Ala Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gly Ala Thr Leu Ala Gly Leu Gly Thr Leu Cys Leu Val
305 310 315 320
Ser Ala Leu Gly Leu Pro Ser Ser Ile Gly Leu Thr Ile Ser Leu Ala
325 330 335
Leu Gly Gly Pro Ala Gly Pro Gly Val Thr Thr Leu Pro Pro Ser Gly
340 345 350
Gly Gly Met Thr Leu Ala Gly Val Ser Leu Thr Cys Leu Val Leu Gly
355 360 365
Pro Thr Pro Ser Ala Ile Ala Val Gly Thr Gly Ser Ala Gly Gly Pro
370 375 380
Gly Ala Ala Thr Leu Thr Thr Pro Pro Val Leu Ala Ser Ala Gly Ser
385 390 395 400
Pro Pro Leu Thr Ser Ala Leu Thr Val Ala Leu Ser Ala Thr Gly Gly
405 410 415
Gly Ala Val Pro Ser Cys Ser Val Met His Gly Ala Leu His Ala His
420 425 430
Thr Thr Gly Leu Ser Leu Ser Leu Ser Leu Gly Leu
435 440
<210> 4
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Val Ile Met Thr Ala Ser Pro Ala Ser Ala Pro Ile Thr Pro Gly
1 5 10 15
Gly Pro Leu Ser Ile Ser Cys Leu Ser Ser Ala Ser Val Ile Thr Ser
20 25 30
Ala Gly Ala Thr Thr Leu Gly Thr Thr Leu Gly Leu Pro Gly Ala Ser
35 40 45
Pro Ala Ala Ala Val Thr Ala Val Ser Ala Ala Pro Ser Gly Val Pro
50 55 60
Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Leu Val
65 70 75 80
Ser Ala Val Gly Ala Gly Ala Val Gly Ile Thr Thr Cys Thr Ala Gly
85 90 95
Ser His Ile Pro Thr Thr Thr Gly Gly Gly Thr Leu Ala Gly Ile Leu
100 105 110
Ala Thr Val Ala Ala Pro Ser Val Pro Ile Pro Pro Pro Ser Ala Gly
115 120 125
Gly Leu Leu Ser Gly Thr Ala Ser Val Val Cys Leu Leu Ala Ala Pro
130 135 140
Thr Pro Ala Gly Ala Leu Val Gly Thr Leu Val Ala Ala Ala Leu Gly
145 150 155 160
Ser Gly Ala Ser Gly Gly Ser Val Thr Gly Gly Ala Ser Leu Ala Ser
165 170 175
Thr Thr Ser Leu Ser Ser Thr Leu Thr Leu Ser Leu Ala Ala Thr Gly
180 185 190
Leu His Leu Val Thr Ala Cys Gly Val Thr His Gly Gly Leu Ser Ser
195 200 205
Pro Val Thr Leu Ser Pro Ala Ala Gly Gly Cys
210 215
<210> 5
<211> 16
<212> PRT
<213> 人体(Homo sapiens)
<400> 5
Gly Ala Val Ala Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala
1 5 10 15
<210> 6
<211> 897
<212> DNA
<213> 人体ApoE2(Homo sapiens)
<400> 6
aaggtggagc aggccgtgga gacagagcct gagcctgagc tgagacagca gaccgagtgg 60
cagagcggcc agaggtggga gctggccctg ggaagatttt gggattacct gagatgggtg 120
cagaccctgt ccgagcaggt gcaggaggag ctgctgtcca gccaggtgac ccaggagctg 180
agggccttga tggacgagac aatgaaggag ctgaaggctt ataagtccga gctggaggag 240
cagctgaccc ccgtggccga ggagacaaga gccaggctgt ccaaggagct gcaagccgcc 300
caggccaggc tgggagctga catggaggat gtgtgcggca ggctggtgca gtacagaggc 360
gaggtgcagg ccatgctggg ccagtccacc gaggagctga gggtgagact ggcctcccac 420
ctgaggaagc tgaggaagag gctgctgaga gacgccgacg atctgcaaaa gtgcctggcc 480
gtgtaccagg ccggcgccag agagggagcc gagagaggac tgtccgccat cagagagaga 540
ctgggacctc tggtggagca gggcagagtg agggccgcca ccgtgggaag cctggctgga 600
cagcctctgc aagagagggc tcaagcctgg ggcgagagac tgagagccag gatggaggag 660
atgggcagca ggaccaggga cagactggac gaggtgaagg agcaggtggc cgaggtgaga 720
gccaagctgg aggagcaagc ccagcagatc aggctgcaag ccgaggcttt tcaggccaga 780
ctgaagagct ggttcgagcc tctggtggaa gacatgcaga gacagtgggc cggcctggtg 840
gagaaggtgc aggccgctgt gggcacaagc gccgctccag tgcctagcga taaccac 897
<210> 7
<211> 897
<212> DNA
<213> 人体ApoE3(Homo sapiens)
<400> 7
aaggtggagc aggccgtgga gacagagcct gagcctgagc tgaggcagca gaccgagtgg 60
cagtccggcc agaggtggga gctggccctg ggaagattct gggattacct gaggtgggtg 120
cagaccctgt ccgagcaggt gcaggaggag ctgctgagca gccaggtgac ccaggagctg 180
agagccctga tggacgagac aatgaaggag ctgaaggctt ataagagcga gctggaggag 240
cagctgacac ccgtggccga ggagacaagg gccagactga gcaaggagct gcaagccgcc 300
caggccagac tgggcgctga catggaggac gtgtgcggca gactggtgca gtacaggggc 360
gaggtgcagg ccatgctggg ccagagcaca gaggagctga gagtgagact ggcctcccac 420
ctgaggaagc tgaggaagag gctgctgagg gatgccgatg acctgcaaaa gagactggcc 480
gtgtaccagg ccggcgccag ggaaggagcc gagagaggac tgtccgccat cagagagagg 540
ctgggacctc tggtggagca gggcagggtg agggccgcta ccgtgggaag cctggccgga 600
cagcccctgc aagagagagc ccaggcttgg ggcgagagac tgagagccag gatggaggag 660
atgggctcca ggaccaggga caggctggat gaggtgaagg agcaggtggc cgaggtgaga 720
gccaagctgg aggagcaagc ccagcagatc aggctgcaag ccgaggcttt tcaggccagg 780
ctgaagagct ggtttgagcc cctggtggag gatatgcaga gacagtgggc cggcctggtg 840
gagaaggtgc aggccgctgt gggcaccagc gccgctcctg ttcctagcga caatcac 897
<210> 8
<211> 897
<212> DNA
<213> 人体ApoE4(Homo sapiens)
<400> 8
aaggtggagc aggccgtgga gacagagccc gagcctgagc tgagacagca gaccgagtgg 60
cagtccggcc agagatggga gctggccctg ggcagattct gggactacct gaggtgggtg 120
cagacactgt ccgagcaggt gcaggaggag ctgctgtcca gccaggtgac ccaggagctg 180
agggccttga tggatgagac aatgaaggag ctgaaggctt ataagtccga gctggaggag 240
cagctgacac ccgtggccga ggagacaaga gccagactga gcaaggagct gcaagccgcc 300
caggccagac tgggcgctga tatggaggat gtgagaggca gactggtgca gtacagaggc 360
gaggtgcagg ccatgctggg ccagagcaca gaggagctga gagtgaggct ggcctcccac 420
ctgaggaagc tgagaaagag actgctgagg gatgccgatg acctgcaaaa gaggctggcc 480
gtgtaccagg ccggcgccag agagggagcc gagagaggac tgtccgccat cagggagaga 540
ctgggacctc tggtggagca gggcagggtg agagccgcca ccgtgggaag cctggccgga 600
caacctctgc aagagagggc tcaagcctgg ggcgagaggc tgagggccag aatggaggag 660
atgggctcca ggacaagaga tagactggac gaggtgaagg agcaggtggc cgaggtgagg 720
gccaagctgg aggagcaagc ccagcagatc agactgcaag ccgaggcttt tcaggccagg 780
ctgaagtcct ggttcgagcc cctggtggag gacatgcaga ggcagtgggc cggcctggtg 840
gagaaggtgc aggccgctgt gggcacatcc gccgctcctg tgccctccga caatcac 897
<210> 9
<211> 299
<212> PRT
<213> 人体ApoE2(Homo sapiens)
<400> 9
Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly
1 5 10 15
Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala
20 25 30
Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly
35 40 45
Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met
50 55 60
Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly
65 70 75 80
Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly
85 90 95
Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Cys
100 105 110
Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly
115 120 125
Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu
130 135 140
Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Cys Leu Ala
145 150 155 160
Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala
165 170 175
Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala
180 185 190
Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly
195 200 205
Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala
210 215 220
Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala
225 230 235 240
Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala
245 250 255
Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met
260 265 270
Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly
275 280 285
Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His
290 295
<210> 10
<211> 299
<212> PRT
<213> 人体ApoE3(Homo sapiens)
<400> 10
Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly
1 5 10 15
Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala
20 25 30
Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly
35 40 45
Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met
50 55 60
Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly
65 70 75 80
Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly
85 90 95
Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Cys
100 105 110
Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly
115 120 125
Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu
130 135 140
Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Ala Leu Ala
145 150 155 160
Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala
165 170 175
Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala
180 185 190
Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly
195 200 205
Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala
210 215 220
Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala
225 230 235 240
Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala
245 250 255
Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met
260 265 270
Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly
275 280 285
Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His
290 295
<210> 11
<211> 299
<212> PRT
<213> 人体ApoE4(Homo sapiens)
<400> 11
Leu Val Gly Gly Ala Val Gly Thr Gly Pro Gly Pro Gly Leu Ala Gly
1 5 10 15
Gly Thr Gly Thr Gly Ser Gly Gly Ala Thr Gly Leu Ala Leu Gly Ala
20 25 30
Pro Thr Ala Thr Leu Ala Thr Val Gly Thr Leu Ser Gly Gly Val Gly
35 40 45
Gly Gly Leu Leu Ser Ser Gly Val Thr Gly Gly Leu Ala Ala Leu Met
50 55 60
Ala Gly Thr Met Leu Gly Leu Leu Ala Thr Leu Ser Gly Leu Gly Gly
65 70 75 80
Gly Leu Thr Pro Val Ala Gly Gly Thr Ala Ala Ala Leu Ser Leu Gly
85 90 95
Leu Gly Ala Ala Gly Ala Ala Leu Gly Ala Ala Met Gly Ala Val Ala
100 105 110
Gly Ala Leu Val Gly Thr Ala Gly Gly Val Gly Ala Met Leu Gly Gly
115 120 125
Ser Thr Gly Gly Leu Ala Val Ala Leu Ala Ser His Leu Ala Leu Leu
130 135 140
Ala Leu Ala Leu Leu Ala Ala Ala Ala Ala Leu Gly Leu Ala Leu Ala
145 150 155 160
Val Thr Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Gly Leu Ser Ala
165 170 175
Ile Ala Gly Ala Leu Gly Pro Leu Val Gly Gly Gly Ala Val Ala Ala
180 185 190
Ala Thr Val Gly Ser Leu Ala Gly Gly Pro Leu Gly Gly Ala Ala Gly
195 200 205
Ala Thr Gly Gly Ala Leu Ala Ala Ala Met Gly Gly Met Gly Ser Ala
210 215 220
Thr Ala Ala Ala Leu Ala Gly Val Leu Gly Gly Val Ala Gly Val Ala
225 230 235 240
Ala Leu Leu Gly Gly Gly Ala Gly Gly Ile Ala Leu Gly Ala Gly Ala
245 250 255
Pro Gly Ala Ala Leu Leu Ser Thr Pro Gly Pro Leu Val Gly Ala Met
260 265 270
Gly Ala Gly Thr Ala Gly Leu Val Gly Leu Val Gly Ala Ala Val Gly
275 280 285
Thr Ser Ala Ala Pro Val Pro Ser Ala Ala His
290 295
Claims (9)
1.一种检测早期老年痴呆症的免疫组化试剂盒，其特征在于，包括以下试剂：
试剂A：APOE4点突变单克隆抗体，
试剂B：HRP标记的二抗工作液，
试剂C：过氧化物酶失活剂，
试剂D：聚合物增强剂，
PBST洗涤液，抗原修复液，DAB显色液及复染工作液；
其中APOE4点突变单克隆抗体重链氨基酸序列如SEQ ID NO：3所示，轻链氨基酸序列如SEQ ID NO：4所示。
2.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，所述APOE4点突变单克隆抗体由保藏于中国典型培养物保藏中心CCTCC，保藏编号为：CCTCC NO：C2020121的杂交瘤细胞株ACE8产生。
3.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，试剂A为3% BSA-PBST配制的APOE4单克隆抗体液体，浓度为0.2μg/μL。
4.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，试剂B为HRP标记的抗鼠IgG聚合物，浓度为1mg/ml。
5.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，试剂C过氧化物酶失活剂为3%H2O2-甲醇溶液。
6.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，试剂D中的聚合物增强剂为PEG4000。
7.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，所述PBST洗涤液为含有体积百分比为0.01% Tween-20的0.01 mol/L PBS缓冲液。
8.根据权利要求1所述的检测早期老年痴呆症的免疫组化试剂盒，其特征在于，所述抗原修复液为10mmol/L的枸橼酸盐缓冲液，pH6.0。
9.权利要求1-8任一项所述的检测早期老年痴呆症的免疫组化试剂盒的使用方法，其特征在于，包括以下步骤：
（1）组织切片脱蜡和水化：二甲苯5 min×2次→无水乙醇5 min×1次→95%乙醇5 min×1次→80%乙醇5 min→70%乙醇5 min→自来水冲洗片刻→PBST洗涤液冲洗1 min×3次；
（2）抗原修复：压力锅中加入足以淹没切片的抗原修复液，加热至沸腾，放入切片，扣上压力阀，继续加热至喷气，维持1.5min，取出自然冷却至室温；
（3）洗涤：ddH2O浸泡5分钟，清洗2次，PBST洗涤液浸泡5分钟，清洗2次；
（4）灭活酶：每片滴加试剂C ，室温处理15min；
（5）洗涤：PBST洗涤液浸泡5分钟，清洗3次；
（6）加一抗：加入试剂A，37℃，湿盒中孵育1小时；
（7）洗涤；PBST洗涤液浸泡5分钟，清洗3次；
（8）加聚合物增强剂：加入试剂D 50μl，湿盒中室温孵育30分钟；
（9）洗涤：PBST洗涤液浸泡5分钟，清洗3次；
（10）加酶标二抗：加入试剂B 工作液，室温37℃，孵育30分钟；
（11）洗涤：PBST洗涤液浸泡5分钟，清洗3次；
（12）显色：每张切片滴加100μl DAB显色液，同时在显微镜下观察控制显色程度，待出现阳性反应，则置流水下冲洗及时终止反应；
（13）复染：将切片放入复染工作液中，染色30min，用蒸馏水冲洗干净；1%盐酸-乙醇分化，流水冲洗；脱水、封片。
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