CN103592439B - 人尿路上皮癌特异性抗体及其应用 - Google Patents
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Abstract
本发明提供了一种新的人尿路上皮癌肿瘤标记物——人肿瘤坏死因子受体超家族TNFRSF25，和产生抗该肿瘤标记物的一株单克隆抗体的杂交瘤细胞，及其分泌的单克隆抗体DTUC1，其是由保藏号为CGMCC NO.8118的杂交瘤分泌而得。单克隆抗体DTUC1与人尿路上皮癌组织及尿路上皮癌细胞系5637、UMUC3、SW780、J82、T24和RT4等呈强阳性反应，而与人正常尿路上皮组织和其它非尿路上皮癌细胞无交叉反应，且经质谱检测，其识别的抗原为TNFRSF 25。本发明还提供了包含单克隆抗体DTUC1的体外诊断试剂盒和使用单克隆抗体DTUC1检测尿液中肿瘤标记物含量的方法。
Description
技术领域
本发明属于肿瘤免疫学领域。具体地说本发明涉及一株抗人尿路上皮癌TNFRSF25的单克隆抗体DTUC1。该抗体能够在生化、细胞和组织水平特异性识别人尿路上皮癌TNFRSF25。本发明还涉及一种双抗夹心酶联免疫吸附检测人尿路上皮癌的方法。这种检测方法中的固定相为B‐8抗体（购自Santa Cruz Biotechnology,Inc.），检测抗体是本发明中的DTUC1，用于检测尿液中肿瘤标记物TNFRSF25含量。
背景技术
尿路上皮癌是指覆盖尿路的上皮细胞发生的肿瘤，包括膀胱尿路上皮癌、输尿管尿路上皮癌和肾盂尿路上皮癌，它们占泌尿系统肿瘤的绝大部分，其中膀胱尿路上皮癌占膀胱癌发生率的90%以上[1]。对尿路上皮肿瘤的诊断，膀胱镜或输尿管镜加病理活检是金标准，但由于膀胱镜或输尿管镜为有创性检查且敏感度不高，对尿路上皮肿瘤的早期诊断和术后监测帮助不大[2]。尿脱落细胞镜检是一种无创的检查，特异度较高，但是敏感度较低，对于尿路上皮肿瘤的早期诊断和术后监测帮助有限[3]。人们一直在寻找一些无创的，敏感度和特异度均高的检查指标来辅助尿路上皮肿瘤的早期诊断和术后监测[4]。
TNFRSF25又名“死亡受体DR3”（Death Receptor3）发现于1996年,为肿瘤坏死因子受体超家族（TNFR super family）中的一员,基因定位在lp36.3，其编码产物为一种11型跨膜蛋白，大小为417个氨基酸，与该家族中其他蛋白类似，其胞质区同样具有一段有蛋白水解功能的氨基酸残基段，即死亡结构域（Death domain），并且与TNFR‐1以及Fas存在高度同源性，通过此区域可使得凋亡信号向通路下游继续传递，继而启动调亡而TNFRSF25同时还可激活NF‐κB的表达，从而使NF‐κB进入细胞核内,激活转录[5]。因其被发现时主要分布于淋巴组织中，如脾脏、胸腺以及外周血淋巴细胞，包括淋巴细胞、NK细胞、巨噬细胞等中[6‐8]，故对其的最初研究多为在免疫调节机制中的作用，并且己证实TNFRSF25可以促进多种炎症性疾病的发生及发展，如炎症性肠病、关节炎等[8‐11]。随着研究的深入，逐渐证实TNFRSF25在多种肿瘤组织中亦有分布[12‐15]。但在尿路上皮癌中，TNFRSF25的高表达鲜有报道。
在尿路上皮癌的诊断中，早期发现和预后判断对临床治疗显得极为重要。近年来用于临床的分子标记，如核基质蛋白、端粒酶和微卫星不稳定性等，其灵敏度或特异性不能令人满意。所以，寻求有效、简便、快速、灵敏的尿路上皮癌的高通量诊断方法是非常必要的。
发明内容
本发明利用人尿路上皮癌组织匀浆免疫小鼠，获得杂交瘤细胞。用ELISA的方法筛选到一株能与人尿路上皮癌组织高度特异结合的抗体DTUC1。免疫组织化学和流式细胞术证实，DTUC1抗体与人尿路上皮癌组织和尿路上皮癌细胞系5637、UMUC3、SW780、J82、T24和RT4呈强阳性反应，而与人正常尿路组织和其它非尿路上皮癌细胞无交叉反应。
本发明还涉及一种双抗夹心酶联免疫吸附检测人尿路上皮癌的方法。这种检测方法中的固定相为B‐8抗体（购自Santa CruzBiotechnology,Inc.）检测抗体是本发明中的DTUC1。
本发明利用DTUC1抗体制备成诊断试剂，即将B‐8抗体（购自Santa Cruz Biotechnology,Inc.）作为固定相，DTUC1抗体检测尿液中肿瘤标记物TNFRSF25含量。
本发明的创新点在于：（1）研制了一株抗人尿路上皮癌的鼠单克隆抗体DTUC1，该组抗体能特异性地结合人尿路上皮癌组织；（2）开发了一种高灵敏度的双抗夹心酶联免疫吸附方法，用于人尿液中尿路上皮癌肿瘤标记物TNFRSF25的检测。
发明详述
本发明提供了一个新的抗人尿路上皮癌TNFRSF25的单克隆抗体的杂交瘤细胞，及其分泌的单克隆抗体DTUC，DTUC1识别TNFRSF25的抗原表位。DTUC1抗体与人尿路上皮癌组织及人尿路上皮癌细胞系5637、UMUC3、SW780、J82、T24和RT4呈强阳性反应，而与人正常尿路组织和其它非尿路上皮癌细胞无交叉反应。本发明还提供了包含单克隆抗体DTUC1体外诊断试剂盒和使用单克隆抗体DTUC1检测尿液中尿路上皮癌肿瘤标记物含量的方法。
本发明的另一个目的是提供一种用于检测或治疗人尿路上皮癌的试剂盒，其包含本发明上述的抗体。
在一个优选实施方案中，在所述的试剂盒中，所述检测是通过酶联免疫吸附法进行，优选所述酶联免疫吸附法是双抗夹心酶联免疫吸附法，其中将B‐8抗体（购自Santa Cruz Biotechnology,Inc.）作为固定相，用DTUC1抗体检测尿液中肿瘤标记物含量。在一个优选实施方案中，所述待测样品是人的尿液。
本发明另一个目的是提供分泌抗人尿路上皮癌TNFRSF25的抗体DTUC1的杂交瘤细胞株，其保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心；保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；保藏日期：2013年08月26日；保藏编号：CGMCC No.8118；建议的分类名名：抗人尿路上皮癌单克隆抗体DTUC1杂交瘤细胞株。
附图说明
图1.DTUC1单抗对人尿路上皮癌组织切片的免疫组化染色；
图2.DTUC1单抗对人正常尿路上皮组织切片的免疫组化染色；
图3.TNFRSF25的氨基酸序列。
具体实施方式
下文将参考实施例详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例1：DTUC1单克隆抗体的制备和纯化
（1）杂交瘤的制备
以人尿路上皮癌组织匀浆免疫Balb/C小鼠（购自北京维通利华实验动物技术有限公司），以100微克/500微升/只的剂量进行腹腔注射。2周后对小鼠进行再次免疫，注射体积和方法不变。待小鼠血清效价达到要求后准备进行细胞融合，融合前三天对小鼠进行加强免疫。在免疫小鼠的同时准备小鼠骨髓瘤细胞SP2/0（ATCC：CRL‐1772）。应用杂交瘤技术（Kohler and Milstein1975）将致敏的B淋巴细胞与骨髓瘤细胞融合，用HAT培养基（购于Invitrogen公司）进行选择性培养。
接着，用ELISA方法检测杂交瘤细胞培养上清：以人尿路上皮癌组织匀浆（1微克/毫升）包被96孔板，每孔100微升，37度培养过夜。加待检上清100微升，37度孵育1小时。洗涤3次，加辣根酶标记的二抗（抗小鼠IgG‐HRP，购自武汉博士德生物工程有限公司），37度孵育1小时。洗涤3次，加TMB（购自武汉博士德生物工程有限公司）50微升显色，室温静置5分钟，加终止液50微升。结果用酶标仪测定波长450nm的OD值，OD值高于阴性对照2倍以上者可视为阳性。
然后，将选出的阳性杂交瘤细胞克隆化培养。经过2‐3轮克隆化培养，获得稳定的能够产生高效价单抗的杂交瘤细胞克隆。将杂交瘤细胞克隆扩大培养，并冻存保种。
本发明中的一种阳性杂交瘤细胞为抗人尿路上皮癌单克隆抗体杂交瘤细胞株，该杂交瘤细胞株于2013年8月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心（CGMCC，中国，北京），保藏号为CGMCC No.8118。
（2）DTUC1单抗的制备和纯化
将上述杂交瘤细胞DTUC1接种至Balb/C小鼠腹腔，制备腹水，再从腹水中提取单抗。单抗DTUC1的纯化：采用Protein G亲和层析法。首先制备Protein G亲和层析柱（购于“GE”公司），用PBS（磷酸盐缓冲液）平衡柱子后，取含DTUC1单抗的腹水过柱，然后用PBS洗柱子，至OD值接近于零，以0.2M的甘氨酸‐HCl溶液（pH2.8）洗脱，收集洗脱液，测定各收集管的OD值，保留峰值区的洗脱液，洗脱液经透析浓缩后20度冻存。
实施例2：DTUC1单抗的鉴定
在实施例1中制备的DTUC1单抗，按常规方法对人尿路上皮癌组织切片（获自中国医学科学院肿瘤医院）进行免疫组化染色，结果如图1、2所示。结果表明，人尿路上皮癌组织经DTUC1单抗、二抗（抗小鼠IgG‐HRP）及DAB底物（武汉博士德生物工程有限公司）染色后呈阳性反应，而人正常尿路上皮组织经DTUC1单抗、二抗及底物染色后呈阴性反应。
用DTUC1单抗，按常规方法对尿路上皮癌细胞系5637、UMUC3、SW780、J82、T24和RT4及其他细胞进行流式细胞仪检测。结果如表1所示。结果表明，DTUC1单抗均与5637、UMUC3、SW780、J82、T24和RT4细胞呈强阳性反应，与其它非尿路上皮癌细胞无交叉反应。表1.流式细胞术检测抗人尿路上皮癌单抗DTUC1对多种细胞的免疫反应
| 细胞系 | DTUC1抗体 |
| 人尿路上皮癌细胞系5637 | ＋ |
| 人尿路上皮癌细胞系UMUC3 | ＋ |
| 人尿路上皮癌细胞系SW780 | ＋ |
| 人尿路上皮癌细胞系J82 | ＋ |
| 人尿路上皮癌细胞系T24 | ＋ |
| 人尿路上皮癌细胞系RT4 | ＋ |
| 人正常尿路上皮细胞系HCV29 | － |
| 人结肠癌细胞系LoVo | － |
| 人宫颈癌细胞系HeLa | － |
| 人肝细胞癌细胞系HepG2 | － |
| 人胚肾细胞系293 | － |
| 人乳腺癌细胞系MCF7 | － |
| 人前列腺癌细胞系PC1 | － |
| 人慢性白血病细胞系K562 | － |
| 人正常外周血单核细胞 | － |
表2.DTUC1抗原的质谱鉴定
| Accession | Mass | Score |
| TNFRSF25HUMAN | 179020 | 128 |
| K2C1HUMAN | 35258 | 37 |
| TFP11HUMAN | 97256 | 37 |
| K1C9HUMAN | 63417 | 35 |
| LRC72HUMAN | 54863 | 35 |
| ALEX HUMAN | 13076 | 35 |
| ATP9B HUMAN | 12919 | 35 |
| UMPS HUMAN | 33255 | 32 |
| K1C10HUMAN | 52637 | 32 |
| SPTN1HUMAN | 35625 | 30 |
| K1C9HUMAN | 15254 | 30 |
由上述可知，本发明的单克隆抗体可以特异性识别尿路上皮癌的抗原，且经质谱检测发现，其特异性识别的抗原为人TNFRSF25。
实施例3：人尿路上皮癌免疫诊断试剂的制备
利用本发明中所述DTUC1抗体，结合抗人TNFRSF25抗体B‐8（购自Santa Cruz Biotechnology,Inc.），开发出了一种高灵敏度的双抗夹心酶联免疫吸附方法，检测人尿液中尿路上皮癌肿瘤抗原。主要方法是用B‐8抗体（购自Santa Cruz Biotechnology,Inc.）作为固定相，辣根过氧化酶（HRP）标记的DTUC1抗体作为检测抗体，检测人尿液中的尿路上皮癌的肿瘤抗原TNFRSF25。实验证实本发明的尿路上皮癌免疫诊断试剂与已有技术相比具有以下积极效果：（1）直接检测尿液，无损伤性。（2）灵敏度高、特异性强，采用双抗夹心酶联免疫吸附法检测细胞中的尿路上皮癌肿瘤抗原，避免了细胞病理学方法本身无法克服的人为性较高和特异性及灵敏度较低的缺点。（3）方便快捷，适合于早期大样本筛查及肿瘤预后的检测。
具体实验方法如下：
1)样品来源及临床资料：健康志愿者和尿路上皮癌患者的尿液均来自中国医学科学院肿瘤医院。
2)具体步骤：
a ELISA板包被抗体：将抗人TNFRSF25抗体B‐8用PBS稀释至1微克/毫升，50微升/孔，4度包被平板过夜。
b 封闭非特异结合位点：200微升/孔的BSA/PBS（2%），37度孵育2小时。
c 样品孵育：同时加入新鲜尿液和辣根酶标记的DTUC1抗体（1微克/毫升）各50微升，37度孵育1小时。健康志愿者的尿液取代样品作为阴性对照。
d PBST洗涤5次。
e 加入辣根酶（HRP）标记的山羊抗小鼠IgG抗体（Sigma公司），100微升/孔，37度孵育1小时。
f PBST洗涤5次。
g 加入底物（四甲基联苯胺，H2O2）100μl/孔，室温避光孵育10‐20分钟；加入2摩尔/升的H2SO450微升中止反应；在酶标仪上测定OD值（450nm）。
3)结果分析：根据阴性OD值/OD临界值≈2.1，得到OD临界值≈阴性OD值/2.1，根据OD临界值，可判断其灵敏度和特异性。
临床灵敏度可用来衡量某种试验检测出有病者的能力，灵敏度是将实际有病的人正确地判定为真阳性的比例。
本实验灵敏度＝92/(92+8)×%＝92%。
临床特异度是衡量试验正确地判定无病者的能力，特异度是将实际无病的人正确地判定为真阴性的比例。
本实验特异度＝95/(5+95)×%＝95%。
尽管本发明的具体实施例已经描述如上，但是可以知道本发明可以进行除了上述说明以外的实践。本发明的保护范围不受说明书的限制。
行除了上述说明以外的实践。本发明的保护范围不受说明书的限制。
参考文献
[1] 王鹏;靳风烁;叶锦;吴刚;李彦锋;张尧;梁平;;荧光原位杂交检测尿脱落细胞染色体异常诊断膀胱肿瘤的研究;第三军医大学学报;2009年10期
[2] 燕翔;丁强;张元芳;孙则禹;徐永华;;尿路上皮癌Survivin的表达及其临床意义;临床肿瘤学杂志;2006年05期
[3] 桂律,许祖德,罗金芳,林梅绥,李如昌;膀胱尿路上皮癌MMP-2表达及其与FAK、p53、bcl-2、Ki-67的关系;临床与实验病理学杂志;2003年01期
[4] 寿建忠,王明荣,马建辉,徐昕,许秉责,吴旻;膀胱移行细胞癌的分子细胞遗传学研究;中华肿瘤杂志;2000年01期
[5] Ashkenazi A,Dixit VM.Death receptor;Signaling andmodulation.Science.1998,28;281(5381):1305-1308.
[6] Croft M.The role of TNF superfamily members in T-ceJlfunction and diseases.Nat.Rev,Immunol.2009;9(4):271-285.
[7] Kang YJ,Kim WJ,Bae HU,et al.Involvement of TL1A and DR3in induction of proinflammatory cytokines and MMP-9 inatherogenesis.Cytokine.2005,7;29(5):229-235.
[8] Fang L,Adkins B,Deyev V,et aLEssential role of TNF receptorsuperfamily25(TNFRSF25)in the development of allergic lunginflammation.J.Exp.Med.2008,12;205(5):1037-1048.
[9] Migone TS,Zhang J,Luo X,et al.TL1A is a TNF-like ligandfor DR3and TR6/DcR3and functions as a T cell costimulator.Immunity.2002,16(3):479-492.
[10] Pappu BP,Borodovsky A,Zheng TS,et al,TL1A-DR3interaction regulates Th17cell function and Thl7-mediatedautoimmune disease.J.Exp.Med2008,12;205(5):1049-1062.
[11] Meylan F,Davidson TS,Kahle E,el al.The TNF-familyreceptor DR3is essential for diverse T cell-mediatedinflammatory diseases.Immunity.2008,18;29(1):79-89.
[12] Bamias G,Martin C,Marini M,et al.Expression,localization,and functional activity of TL1A,a novel Th1polarizing cytokine in inflammatory disease.J,Immunol.2003,1;171(9):4868-4874.
[13] Warzocha K,Ribeiro P,Chariot C,et al.A new death receptor3isoform:expression in human lymphoid cell lines andnon-Hodgkin's lymphomas.Biochem Biophys ResCommun.1998,14;242(2):376-379.
[14] Gout S,Morin C,Houle F,et al.Death receptor-3,a newE-Selectin counter-receptor that confers migration andsurvival advantages to colon carcinoma cells by triggering p38and ERK MAPK activation.Cancer Res.2006,15;66(18):9117-9124.
[15] Jiang S,Song MJ,Shin EC,et al.Apoptosis in human hepatomacell lines by chemotherapeutic drugs via Fas-dependent andFas-independent pathways.Hepatology.1999;29(1):101-10.
<110> 李翀
<120> 人尿路上皮癌特异性抗体及其应用
<160> 1
<210> 1
<212> protein
<213> 人尿路上皮癌特异性抗体及其应用
<400> 1
MEQRPRGCAAAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPC 60
TEPCGNSTCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADTRCGCKPGW 120
FVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPT 180
PPPSLAGAPWGAVQSAVPLSVAGGRVGVFWVQVLLAGLVVPLLLGATLTYTYRHCWPHKP 240
LVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEAL
300
CPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFV 360
RTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRS 420
RLQRGP 426
Claims (3)
1.一种人肿瘤坏死因子受体超家族TNFRSF25的抗原表位在制备检测人尿路上皮癌试剂盒中的应用，其特征在于所述TNFRSF25的抗原表位氨基酸序列为SEQ ID NO:1所示，其中检测的样本为人尿路上皮组织。
2.根据权利要求1所述的应用，其特征在于所述试剂盒包含单克隆抗体DTUC1，该单克隆抗体由保藏号为CGMCC No.8118的杂交瘤细胞株分泌。
3.根据权利要求2所述的应用，其中所述检测是通过酶联免疫吸附法进行，所述酶联免疫吸附法是双抗夹心酶联免疫吸附法。
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Effective date of registration: 20220808 Address after: 101400 floor 1-4, building 1, yard 9, Yanqi street, Yanqi Economic Development Zone, Huairou District, Beijing Patentee after: Zhongke Jianlan (Beijing) Medical Research Institute Address before: 570100 106-1, Zone E, Fuxing City, 32 Binhai street, Longhua District, Haikou City, Hainan Province Patentee before: Hainan Mingyi Cuisine Technology Co.,Ltd. |
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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151021 |
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| CF01 | Termination of patent right due to non-payment of annual fee |