CN104792998B - Loxl2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用 - Google Patents
Loxl2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用 Download PDFInfo
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Abstract
本发明提供了LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。LOXL2的表达与甲状腺癌预示、侵袭、转移、预后有相关性,不仅可作为甲状腺癌相关试验研究中癌组织的预示、侵袭、转移标志物的应用,而且可以提高病理早期预示甲状腺癌的灵敏度。因LOXL2为分泌蛋白,还可作为非侵入性的蛋白标志物,在甲状腺癌预示、疗效评估、复发监控等方面有应用的前景。
Description
技术领域
本发明涉及医学生物技术领域的抗体,具体的说,是赖氨酰氧化酶样蛋白2抗原用于制备甲状腺癌预示、侵袭、转移检测用试剂盒的应用。
背景技术
甲状腺癌发病率居头颈部肿瘤之首,近年来对甲状腺癌组织的预示、侵袭、转移标志物相关试验逐渐成为研究的热点。
发明内容
针对现有技术的缺陷或不足,本发明提供了LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。
进一步提供LOXL2抗原、LOXL2单抗和LOXL2多抗用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。
本发明的试剂盒为ELISA试剂盒。
同时还提供了一种用于甲状腺癌预示、侵袭、转移检测试剂盒,该试剂盒包括LOXL2抗原。
进一步提供的试剂盒包括:LOXL2抗原、LOXL2单抗和LOXL2多抗。
本发明的LOXL2抗的氨基酸序列为:
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
申请人首次发现LOXL2的表达与甲状腺癌预示、侵袭、转移、预后有相关性,不仅可作为甲状腺癌相关试验研究中癌组织的预示、侵袭、转移标志物的应用,而且可以提高病理早期预示甲状腺癌的灵敏度。因LOXL2为分泌蛋白,还可作为非侵入性的蛋白标志物,在甲状腺癌预示、疗效评估、复发监控等方面有应用的前景。
附图说明
图1是实施例1中的癌与癌旁Ratio分布图;
图2是实施例2的ROC曲线下面积;
图3是实施例3的ROC曲线下面积;
图4LOXL2蛋白相互作用预测网络图谱;Cytoscape用于分子网络预测,945066为LOXL2,947814为TP53,946724为CA125,947803为CEA,944821为HER2,945672为BRCA1,948490为BRCA1,946234为cystatinD,947429为ER,851539为PR。
以下结合附图和实施例对本发明作进一步的详细说明。
具体实施方式
赖氨酰氧化酶样蛋白2(Lysyloxidaselikeprotein2,LOXL2)是赖氨酰氧化酶(Lysyloxidase,LOX)家族中的一种依赖铜的分泌型胺氧化酶,催化细胞外基质胶原与弹力蛋白交联,使其纤维化,在保持细胞外基质结构的完整性、细胞运动、肿瘤发生发展等生物学功能方面起重要作用。
为了证明LOXL2在甲状腺癌预示、侵袭、转移标志物的实际应用,申请人采用Real-timePCR(染料法、探针法)检测甲状腺穿刺组织中LOXL2mRNA表达量,根据cutoff值,判断病理组织良恶性,辅助病理预示;并采用夹心法ELISA检测血液(血清、血浆)中LOXL2分泌蛋白含量,用于甲状腺癌预示、疗效评估、复发监控等方面的具体试验。
在以下的实施例中,缩略语、英文和关键术语定义列表:
LOXL2 | Lysyloxidase like protein 2 |
CP | Cross point |
GAPDH | glyceraldehyde-3-phosphate dehydrogenase |
PBS | 磷酸盐缓冲液 |
PBST | 磷酸盐吐温缓冲液 |
TMB | 3,3',5,5'-四甲基联苯胺 |
实施例1:Real-timePCR检测甲状腺穿刺组织中LOXL2mRNA表达量(染料法)
设计LOXL2跨外显子的引物,扩增子139bp:
上游引物:CATGGGCTGCTGGGACATG
下游引物:GTTGTTGGAGTAATCGGATTCTGC
GAPDH上、下游引物:
上游引物:GGGTGTGAACCATGAGAAGT
下游引物:GACTGTGGTCATGAGTCCT
使用商品化的新鲜组织RNA提取试剂TRIzolReagent,Invitrigen(货号:15596026),提取的基因组RNA,取1ugRNA逆转录为cDNA(Fermentas商品化试剂盒,货号K1622),稀释10倍后,取2ul作为PCR模板。
上、下游引物终浓度250nM(英潍捷基上海合成),TaqDNA聚合酶(罗氏,货号12032953001),终浓度1U/反应。UDG(NEB),0.5U/反应。
dNTP(TAKARA)终浓度250nM,EvaGREEN1ul/反应。
PCR仪:Roche480。
反应条件:37度,2分钟;预变性95度,5分钟;扩增95度,10秒,60度,15秒,72度,20秒,40个循环。溶解95度,1分钟,40度,1分钟,65度,1秒,95度,持续,每度采集25次信号。
22例癌与癌旁配对样本,相对定量方法,2-ΔΔCP。LOXL2表达水平在癌中明显上调。癌与癌旁Ratio分布图参见图1。
实施例2:Real-timePCR检测甲状腺穿刺组织中LOXL2mRNA表达量(探针法)
LOXL2引物、探针:
上游引物:TCGCCGCCAACGCCACAACCA
下游引物:CGCATCGCTGCTCCCCGAAGAGC
探针:FAM-ACCGCCATGACATCGACTGCCAGTG-TAMRA
使用商品化的新鲜组织RNA提取试剂TRIzolReagent,Invitrigen(货号:15596026),提取的基因组RNA,取1ugRNA逆转录为cDNA(Fermentas商品化试剂盒,货号K1622),稀释10倍后,取2ul作为PCR模板。
上、下游引物终浓度250nM(英潍捷基上海合成),TaqDNA聚合酶(罗氏,货号12032953001),终浓度1U/反应。UDG(NEB),0.5U/反应。
dNTP(TAKARA)终浓度250nM;探针终浓度250nM。
PCR仪:Roche480。
反应条件:37度,2分钟;预变性预变性95度,5分钟;扩增95度,10秒,60度,30秒,45个循环。
合成LOXL2的DNA序列为:
Gccacaaggccagcttctgcttggaggacacagaatgtgaaggagacatccagaagaattacgagtgtgccaacttcggcgatcagggcatcaccatgggctgctgggacatgtaccgccatgacatcgactgccagtgggttgacatcactgacgtgccccctggagactacctgttccaggttgttattaaccccaacttcgaggttgcagaatccgattactccaacaacatcatgaaatgcaggagccgctatgacggccaccgcatctggatgtacaactgccacataggtggttccttcagcgaagagacggaaaaaaagtttgagcacttcagcgggctctta
LOXL2全长氨基酸序列:
LOXL2的氨基酸序列是:
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP
反转录为RNA,作为标准品,106,105,104,103,102,10拷贝,与样本一起扩增,做标准曲线,从曲线上读出样本的拷贝数。
30例甲状腺癌样本,30例炎症、良性增生等样本,统计cutoff值705copy,灵敏度70.4%,特异性78.9%。
ROC曲线下面积:74.3%(图2)。
ROC曲线下的面积值在1.0和0.5之间。在AUC>0.5的情况下,AUC越接近于1,说明诊断效果越好。AUC在0.5~0.7时有较低准确性,AUC在0.7~0.9时有一定准确性,AUC在0.9以上时有较高准确性。AUC=0.5时,说明诊断方法完全不起作用,无诊断价值。AUC<0.5不符合真实情况,在实际中极少出现。
实施例3:双抗夹心酶联免疫吸附(ELISA试剂盒)预示甲状腺癌
该实施例所用试剂为:
LOXL2抗原、LOXL2单抗、LOXL2多抗均购自NovusBiologicals,货号分别为H00004017-Q01、H00004017-M05、NBP1-32954。
多抗标记:用HRP标记兔抗LOXL2多抗,HRP为德国BoehringerMannheim公司产品。
辣根过氧化物酶(HRP),市售。
TMB(3,3',5,5'-四甲基联苯胺),市售。
PBST(磷酸盐吐温缓冲液),市售。
PBS(磷酸盐缓冲液),市售。
检测方法:
采用过硫酸钠法,抗体和辣根过氧化物酶(HRP)的重量比例为1:1,HRP和抗体混合后置4℃条件下过夜,然后以0.02mol/L、pH7.4的PBS于4℃透析过夜,其他采用常规标记方法。
双抗夹心酶联免疫吸附(ELISA试剂盒):0.05mol/L、pH9.6的碳酸盐缓冲液稀释包被抗体至包被浓度,加入酶标板(Corning)100μl/孔,置4℃条件过夜,甩干。
含30%小牛血清的PBST(其中含0.05%的Tween220)0.02mol/L,pH7.4,封闭200μl/孔,37℃,1小时,甩干,以PBST洗板1次;
加入样品稀释液(含10%的Trinton2100)5μl/孔,然后加入待检测血清50μl/孔,设阴、阳对照各3孔,37℃,30分钟,以PBST冲洗5次,甩干;
将HRP标记的抗体稀释后加入酶标板(50μl/孔),37℃,30分钟,以PBST冲洗5次,甩干;加入底物液和TMB各50μl/孔,37℃,10分钟;
加入2mol/L的H2SO4(50μl/孔),终止反应,于450nm测吸光度(A值)。
LOXL2单抗作为捕获抗体,包被单抗3ug/孔,HRP标记多抗1:1000(抗体浓度与整个试剂浓度之比)稀释,抗原标准品50pg/ml,100pg/ml,200pg/ml。
该实施例检测甲状腺患者血清30例,正常献血员血清30例,当cutoff值取86.3920pg/ml时,检测的灵敏度75.9%,特异性96.7%。ROC曲线下面积0.947(图3)。
实施例4:ELISA甲状腺癌疗效评估
LOXL2的ELISA操作同实施例3,用药前检测血清LOXL2水平,跟踪随访10位患者,疗程结束后再行检测LOXL2水平,对比LOXL2血清含量变化与治疗效果是否有相关性。结果见表1。
表1
由上表可以看出,检测LOXL2血清水平可对甲状腺癌治疗效果进行评估。
实施例5:ELISA甲状腺癌复发监控
LOXL2的ELISA操作同实施例3,对达到治疗终点的患者10例,检测LOXL2血清水平,每半年检测一次,跟踪随访2年,结果如下表2:
表2
Claims (5)
1.LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用,所述的LOXL2抗原的氨基酸序列为:
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
2.LOXL2抗原、LOXL2单抗和LOXL2多抗用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用,所述的LOXL2抗原的氨基酸序列为:
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
3.如权利要求2所述的应用,其特征在于,所述的试剂盒为ELISA试剂盒。
4.一种用于甲状腺癌预示、侵袭、转移检测试剂盒,其特征在于,该试剂盒包括LOXL2抗原,所述的LOXL2抗原的氨基酸序列为:
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
5.如权利要求4所述的试剂盒,其特征在于,所述试剂盒包括:LOXL2抗原、LOXL2单抗和LOXL2多抗。
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