CN115074342B - 一种抗人酸性磷酸酶单克隆抗体及其应用 - Google Patents
一种抗人酸性磷酸酶单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种由合成的人酸性磷酸酶抗原表位肽制备的抗原，来自人溶酶体酸性磷酸酶ACP2的多肽免疫小鼠，通过筛选得到一种小鼠抗人ACP2单克隆抗体，此抗体应用于免疫细胞化学染色或免疫组织化学染色，此染色方法用于宫颈脱落细胞、组织样本的染色，以帮助观察异常鳞状细胞和筛查宫颈癌及癌前病变；以及此方法用于区分恶性浆膜腔积液中间皮细胞和恶性肿瘤细胞，为宫颈癌筛查及恶性浆膜腔积液中间皮细胞和恶性肿瘤细胞区分提供了一种快速、简便、易行且有效的诊断途径。
Description
技术领域
本发明涉及多肽化学和免疫学领域，特别是涉及一种抗人酸性磷酸酶单克隆抗体及其应用。
背景技术
采用巴氏染色方法通过宫颈细胞涂片进行宫颈癌筛查是发达国家最有效和最具成本效益的癌症筛查方法，可降低宫颈癌的发病率和死亡率。然而，宫颈涂片细胞学筛查有显著的假阳性和假阴性率。为了进一步提高使用巴氏涂片进行细胞学筛查宫颈癌前病变的检出率，已经开发了许多辅助方法及检查，包括薄层细胞学检查；使用放大的化学发光筛查检查(窥镜检查)与巴氏涂片相结合；细胞学和宫颈造影联合使用；自动复筛方法；通过杂交捕获或聚合酶链反应测试检测人乳头状瘤病毒DNA；通过端粒重复扩增方案(TRAP)、傅里叶变换红外(FTIR)光谱和p16INK4a蛋白的免疫细胞化学检测来评估端粒酶的重复活性。然而，由于这些方法成本高得令人望而却步，这些新技术的可用性有限，在发展中国家的大规模筛查计划中没有任何作用。
最近有研究发现酸性磷酸酶在发育不良的宫颈细胞中具有高活性。酸性磷酸酶(cervical acid phosphatase，CAP)是一类溶酶体酶的总称，CAP不存在于女性正常生殖道的鳞状细胞中。子宫颈的癌前病变出现在含有CAP的转化区。因此，低级别和高级别鳞状上皮内病变(分别为LSIL和HSIL)的异常细胞均呈CAP阳性。其他病变如 ASC-H(非典型鳞状细胞不能排除高级别SIL)和其他宫颈上皮内瘤变(AGC)病变也表达CAP。非典型鳞状细胞(ASC，根据宫颈TBS报告系统)是指形态上提示为鳞状上皮内病变的细胞改变，但从质量和数量上又不足以做出明确判断。ASC不是一个单一的生物学实体；它包括与致瘤型人类乳头瘤病毒感染无关的改变(炎性)、瘤变，也包括那些提示可能有潜在宫颈上皮内瘤变(CIN)的所见，以及极少数的癌。这也正是目前细胞形态学诊断无法解决的问题，此时若能联合宫颈酸性磷酸酶检测，并能将有瘤变倾向的鳞状上皮细胞标记、区分开来，将会对临床诊断和治疗起到重要的作用，也是宫颈癌筛查细胞学诊断中的重大进步。
浆膜腔是浆膜壁层和脏层之间的间隙，分为胸膜腔、腹膜腔、心包腔等。腔内有少许浆液，起润滑作用，病理情况下可增多，形成胸水、腹水、心包积液等。临床送检的浆膜腔积液均属病理性，浆膜腔积液内的细胞成分包括间皮细胞、非上皮来源的血液细胞，如果存在肿瘤细胞，则为称恶性浆膜腔积液。良性病变(如肺炎、肺梗死、流感、肝脓肿、结核病、心衰等等)或恶性病变(肺癌、乳腺癌、恶性淋巴瘤、恶性间皮瘤、卵巢癌、宫颈癌、子宫内膜癌、消化系统肿瘤等等)都可以引起浆膜腔积液。
世卫组织最新数据显示，肺癌已成为中国癌症死因之首，同时也是中国男性最常见的癌症。约50％以上的肺癌患者在疾病过程中将出现胸水。9年来前六位的肿瘤分别是：肺癌、肝癌、胃癌、食道癌、结直肠肛门癌、乳腺癌，占所有报告病例的65％以上。引起恶性腹水的原发病以卵巢癌和肝癌最常见，占30％～54％，其他可由胃肠道肿瘤、胰腺癌、子宫癌等引起，10％～15％胃肠道肿瘤可发生恶性腹水，腹腔外恶性肿瘤如乳腺癌、肺癌和淋巴瘤等也可引起。此外，15-20％的患者原发病灶不明。而临床的随机胸腹水样本中，25％左右为恶性。
脱落细胞学检查因具有快速、简便、易行、可重复检查以及患者痛苦少等优点，在临床上得以广泛应用，目前是恶性胸腹水诊断的基本方法。细胞学检查依据细胞的形态学特征作出判断，而浆膜腔中的间皮细胞由于增生、变性等原因导致形态多样，仅通过巴氏染色从形态上不容易与癌细胞区分，所以浆膜腔积液的脱落细胞学检查经常存在较低的敏感性(40％-60％)，经验不足的病理医生甚至无法做出判断。因此如何减少浆膜腔积液细胞学诊断的假阴性、假阳性率，避免漏诊、误诊，提高阳性率具有重要的实际意义，这也是目前亟待解决的问题。
发明内容
本发明所要解决的技术问题是提供一种抗人酸性磷酸酶单克隆抗体及其应用，为宫颈癌筛查细胞学诊断及浆膜腔积液细胞学诊断带来了重大的进步。
为解决上述技术问题，本发明采用的第一个技术方案是：提供一种人酸性磷酸酶抗原表位肽，所述人酸性磷酸酶抗原表位肽的氨基酸序列为QNETRQTPEYQN，序列来自人酸性磷酸酶ACP2(Human Lysosomal Acid Phosphatase Isoform 1，Accession No.AAH03160，166-177aa)。
为解决上述技术问题，本发明采用的第二个技术方案是：提供一种人酸性磷酸酶抗原，其是通过如上所述的人酸性磷酸酶抗原表位肽与载体蛋白偶联制备而成的。
为解决上述技术问题，本发明采用的第三个技术方案是：提供一种抗人酸性磷酸酶单克隆抗体，其是由如上所述的人酸性磷酸酶抗原制备而成的单克隆抗体。
为解决上述技术问题，本发明采用的第四个技术方案是：提供一种如上所述的抗人酸性磷酸酶单克隆抗体在免疫细胞化学染色或/和免疫组织化学染色中的应用。
在本发明一个较佳实施例中，所述应用用于宫颈脱落细胞或/和组织样本的染色，以帮助观察异常鳞状细胞和筛查宫颈癌及癌前病变。
在本发明一个较佳实施例中，所述应用用于区分恶性浆膜腔积液中间皮细胞和恶性肿瘤细胞。
本发明的有益效果是：本发明由合成的人酸性磷酸酶抗原表位肽制备的抗原筛选出抗人酸性磷酸酶单克隆抗体，可应用于免疫细胞化学染色或免疫组织化学染色，此染色方法不仅能将有瘤变倾向的鳞状上皮细胞标记、区分开来，将会对临床诊断和治疗起到重要的作用，也是宫颈癌筛查细胞学诊断中的重大进步，而且能够解决目前巴氏染色存在的缺陷及浆膜腔积液细胞学诊断的假阴性、假阳性率高的问题，避免漏诊、误诊，提高阳性率，为宫颈癌筛查及恶性浆膜腔积液中间皮细胞和恶性肿瘤细胞区分提供了一种快速、简便、易行且有效的诊断途径。
附图说明
图1是本发明实施例2宫颈脱落细胞免疫细胞化学染色实验示意图，其中①正常鳞状上皮细胞，ACP2阴性表达；②鳞化细胞，ACP2阳性表达；③ASC-US细胞ACP2 阳性表达(箭头所指)；④ASC-US细胞ACP2阴性表达(组织学鉴定为炎症)；⑤低鳞状上皮内病变细胞ACP2阳性表达(箭头所指)；⑥高鳞状上皮内病变细胞ACP2阳性表达(箭头所指)；⑦鳞状细胞癌ACP2阳性；
图2是本发明实施例5宫颈组织样本免疫组织化学染色(IHC)实验示意图，其中①宫颈鳞癌阳性；②正常鳞状上皮阴性；③腺上皮或伴鳞化阳性，少许基底层细胞可弱阳性；
图3是本发明实施例6恶性浆膜腔积液中间皮细胞与恶性细胞染色实验中腹水脱落细胞染色示意图；
图4是本发明实施例6恶性浆膜腔积液中间皮细胞与恶性细胞染色实验中胸水脱落细胞染色示意图。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域技术人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
下文提到染色，除非指明是巴氏染色，否则全部都是免疫细胞化学染色或免疫组织化学染色。
实施例1：
抗原制备：合成用于制备单克隆抗体的抗原肽序列为QNETRQTPEYQN，序列来自人ACP2(Human Lysosomal Acid Phosphatase Isoform 1，Accession No.AAH03160， 166-177aa)，与BSA(牛血清白蛋白)偶联。
免疫动物：选用6-8周龄雌性Balb/c小鼠。
免疫方案：首先2次剂量50μg，每次间隔3周；加强免疫4次剂量200μg/次，每次间隔1天。首次免疫使用完全佐剂，第二次起使用不完全佐剂。末次加强免疫后第3 天取脾进行融合，制备小鼠脾淋巴细胞。将血清分离后作为抗体检测时阳性对照血清。骨髓瘤细胞定期用8-氮鸟嘌呤处理，使生存的细胞对HAT(次黄嘌呤，hypoxantin；氨基蝶呤，aminopterin；和胸腺嘧啶脱氧核苷，thymidin)呈均一的敏感性。脾细胞和骨髓瘤细胞经聚乙二醇(PEG)处理融合，在HAT选择培养液中生长，筛选出杂交瘤细胞。选取20个单克隆抗体，ELISA方法从中选取效价高且稳定的5个单克隆抗体用于免疫细胞化学染色。
实施例2：宫颈脱落细胞免疫细胞化学染色
1.1样本
临床液基细胞学样本保存于液基细胞保存液中，经液基细胞制片术制片，得到符合临床要求的细胞片。以巴氏染色和免疫细胞化学染色后用于阅片。
1.2样本染色步骤及镜检阅片
液基制片得到的细胞片或涂片95％酒精固定；PBS洗5分钟2次；加一抗稀释液封闭，室温孵育30分钟；弃掉液体，加过氧化氢孵育10分钟；滴加一抗，抗人ACP2，室温条件下孵育1小时；PBS洗3次；滴加HRP-二抗30分钟；PBS洗3次；滴加DAB 显色5分钟；PBS终止显色；苏木素复染；封片后镜检。
染色以鳞状上皮化生或宫颈腺上皮细胞为阳性对照，用抗体的孵育液作阴性对照。有经验的且能熟练掌握判读标准医师进行判读，同时采用随机双盲法，阳性结果的判定标准为光学显微镜下，酸性磷酸酶阳性表现为细胞浆棕色颗粒状，在阳性对照的鳞状上皮化生细胞中，显示丰富的酸性磷酸酶，正常的鳞状上皮、表层细胞中呈阴性，宫颈腺上皮细胞细胞浆中显示均匀的棕色颗粒，在萎缩性反应中，一些底层细胞可呈浅棕色着色，同时组织细胞也可以显色。
1.3诊断标准
采用TBS分类系统作为诊断标准。TBS报告主要包括：(1)良性细胞学改变，主要包括感染(滴虫感染，霉菌性感染，形态学似嗜血杆菌感染，形态学似放射菌感染，形态学似HPV感染，形态学似单纯疱疹病毒感染)和炎症；(2)上皮细胞异常改变：①腺上皮细胞改变，分为没有明确诊断意义的不典型腺体上皮细胞和宫颈腺癌、子宫内膜癌以及其他部位的腺癌；②鳞状上皮细胞异常，分为①不典型鳞状上皮细胞(ASC)：无明确诊断意义的不典型鳞状上皮细胞(ASC-US)和不典型鳞状上皮细胞不除外高度鳞状上皮内病变(ASC-H)；②低度鳞状上皮内病变(LSIL)；③高度鳞状上皮内病变 (HSIL)；④鳞状细胞癌(SCC)。CAP阳性是指ASC-US以上病变。在ACP2染色的涂片中，正常的宫颈管细胞和少数单核细胞为阳性，这些细胞可以根据其形态进行排除。只有ACP2阳性的未成熟的鳞状上皮细胞、核增大的鳞状上皮细胞以及核有异型的鳞状上皮细胞才确定为ACP2染色阳性的细胞。
染色结果：
以正常鳞状上皮ACP2阴性(见图1①)，可作为细胞学染色的阴性对照；以鳞状上皮化生细胞作为阳性对照，细胞内明显有棕色沉淀，为ACP2阳性(见图1②)；ASC-US (巴氏染色判断为ASC-US)细胞中ACP2阳性细胞内有明显棕色沉淀细胞，ACP2阴性细胞无显著沉淀(见图1③～④)；LSIL患者细胞和HSIL患者细胞中ACP2表达均为阳性，有大片棕色沉淀在视野中(见图1⑤～⑥)；鳞癌细胞ACP2表达为阳性(图1 ⑦)。
实施例3：非典型鳞状上皮细胞(ASC-US)进行明确诊断
采用经过细胞学TBS报告系统诊断为ASC-US的妇科宫颈标本，用沉降式液基细胞制片机制备薄层细胞片备用；采用Thermo自动免疫组织化学染色机进行染色，用 ACP2染色结果与组织病理学结果进行比较。
134例经宫颈细胞学检查被判断为未明确意义的非典型鳞状上皮细胞妇科宫颈标本经组织病理学检查后发现，发生炎症改变76例，LSIL50例，HSIL7例，鳞状细胞癌 1例。酸性磷酸酶染色发现，酸性磷酸酶在LSIL，HSIL、鳞状细胞癌中均大量表达，在大部分ACP2表达呈阳性，且差异具有统计学意义(P＜0.05)。
表1组织病理学结果与对应酸性磷酸酶检测结果
实施例4：宫颈癌及癌前病变筛查
1476例宫颈脱落细胞随机样本保存于95％乙醇中，每例样本经液基制片得到2张合格的细胞片，分别进行巴氏染色和ACP2免疫细胞化学染色。有经验的且能熟练掌握判读标准医师按前述标准进行判读，巴氏染色片按TBS系统判读。巴氏染色读片结果、 ACP2染色读片结果分别与组织病理结果比较(表2-表4)。ACP2染色在敏感性、特异性、阳性预测值、阴性预测值及准确性上全面占优。
表2酸性磷酸酶检查结果
表3巴氏染色检查结果
表4诊断评估指标比较
实施例5：宫颈组织样本免疫组织化学染色(IHC)
参阅图2，宫颈组织标本经ACP2免疫组织化学染色后显微镜下观察到：
1)宫颈鳞癌样本，肿瘤细胞排列紊乱，细胞及其核的异形性明显，可见大量病理性核分裂象，ACP2染色阳性(细胞浆内出现棕黄色颗粒)；2)正常鳞状上皮：鳞状上皮细胞阴性，基底层细胞可有弱阳性表达；3)宫颈腺上皮细胞及鳞化细胞阳性，少许基底层细胞可弱阳性。
实施例6：恶性浆膜腔积液
染色方法：剧烈震荡浆膜腔积液，使细胞团分散；取浆膜腔积液1-5ml制作液基细胞片或涂片，室温风干后立即4％多聚甲醛或福尔马林固定。PBS洗5分钟2次；加一抗稀释液室温孵育30分钟；弃掉液体，加过氧化氢孵育10分钟；滴加一抗(按照1： 150稀释比例)，室温条件下孵育30分钟；PBS洗3次；滴加HRP二抗30分钟；PBS 洗3次；滴加DAB(1毫升加1滴)5分钟；PBS终止显色；苏木素复染；经冲洗、脱水后封片。
镜检结果：多聚甲醛固定后红细胞破裂，细胞染色片上无红细胞影响，背景干净。间皮细胞和吞噬细胞酸性磷酸酶表达强阳性，胞浆内含有大量棕褐色染色颗粒；浆膜腔积液中恶性细胞(恶性肿瘤细胞)ACP2染色阴性，胞浆中无染色颗粒。可以清晰鉴别出恶性细胞与非恶性细胞。图3为腹水脱落细胞染色图，箭头指向肿瘤细胞，其胞浆无染色；空心箭头指向间皮细胞，胞浆有棕色染色颗粒。同一病例组织学鉴定为肝癌。图 4为胸水脱落细胞染色图，箭头指向肿瘤细胞团，其胞浆无染色；空心箭头指向间皮细胞团，胞浆有棕色染色颗粒。同一病例组织学鉴定为肺癌。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效形式变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (2)
1.一种人酸性磷酸酶抗原表位肽，其特征在于，所述人酸性磷酸酶抗原表位肽的氨基酸序列为QNETRQTPEYQN，序列来自人酸性磷酸酶ACP2。
2.一种人酸性磷酸酶抗原，其特征在于，其是通过权利要求1所述的人酸性磷酸酶抗原表位肽与载体蛋白偶联制备而成的。
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| CN105424448A (zh) * | 2015-11-06 | 2016-03-23 | 合肥锦慈康生物技术有限公司 | 用于区分恶性浆膜腔积液中间皮细胞与癌细胞的酸性磷酸酶染色法及其应用 |
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| CN105424448A (zh) * | 2015-11-06 | 2016-03-23 | 合肥锦慈康生物技术有限公司 | 用于区分恶性浆膜腔积液中间皮细胞与癌细胞的酸性磷酸酶染色法及其应用 |
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