CN104356226A - 一种检测血浆免疫标志物-vegfr1自身抗体的抗原多肽及应用 - Google Patents
一种检测血浆免疫标志物-vegfr1自身抗体的抗原多肽及应用 Download PDFInfo
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Abstract
本申请提供了一种VEGFR1抗原多肽，其包含H-DEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLE-OH、H-DLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSI-OH和H-ESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAEL-OH三种多肽，及其用于检测血浆免疫标志物-VEGFR1自身抗体的应用。还提供了一种利用本申请所述的VEGFR1抗原多肽检测血浆免疫标志物-VEGFR1自身抗体的方法，以及包含所述VEGFR1抗原多肽的测试盒。
Description
技术领域
本发明属于免疫学技术领域，涉及一种VEGFR1抗原多肽。应用该抗原多肽可制备检测人血浆中VEGFR1抗体水平的特异试剂。
背景技术
近年来的研究表明，发现和测定健康人群血液中的肿瘤相关抗体具有指导靶向治疗价值。如已在临床广泛应用的曲妥株单抗(Trastuzumab)和贝伐株单抗(Bevacizumab)就是以上皮细胞生长因子二型受体(HER2)和血管内皮细胞生长因子A(VEGF-A)为标靶，成为临床治疗晚期肿瘤的重要手段。然而，由于副作用太大，这两种单克隆抗体只能做为三线治疗药物。因此，发展可用于防止肿瘤局部治疗后转移和复发、且低副作用的新型手段是目前亟待解决的问题。而在免疫治疗领域探索解决这个难题的前置技术障碍是找到一种创新性的标靶技术实用方案，然后才能进一步发展全新的肿瘤免疫治疗方案。
VEGFR1也被称为血管内皮细胞生长因子一型受体，已被公认为是癌细胞表达的特异性蛋白之一。经大量的研究发现，许多人类实体肿瘤可大量表达VEGFR1，如肝癌，肺癌，肾癌，肠癌及乳腺癌等。因为VEGFR1抗原表位多肽主要位于肿瘤细胞表面，可直接作为相应抗体杀伤肿瘤的靶点，因此学界公认VEGFR1相应抗体的临床应用极有可能成为最快实现的、低副作用的全新抗癌治疗手段。目前生物技术领域仅有的方法，是以重组蛋白为抗原实施VEGFR1相应抗体的检测和筛选，其过程要经过载体构建、转染、表达、筛选、纯化等繁琐的过程，但由于蛋白空间结构复杂，线性抗原表位不易暴露，因而以重组 蛋白检测出的抗体特异性差，有相当比例无法与肿瘤细胞膜表面的抗原靶点结合，因而仅能进行低标准的定性检测手段，无法据此发展用于防治肿瘤的实用手段。同时，ELISA法的高度灵敏性对蛋白纯化技术的稳定性要求极高，因此符合检测要求的重组蛋白成本昂贵。国内外至今尚无有关VEGFR1相应抗体检测和筛选的有效手段。
本发明针对性设计的线性VEGFR1抗原表位多肽能有效解决上述技术难题，能制备特异性极高的检测试剂，按设定的流程规范检测免疫标志物-VEGFR1抗体在健康献血者血浆中的浓度指标值，可获得精度高、操作简易、成本适中的VEGFR1抗体检测、甄别、定性和定量应用的一揽子解决方案。因而本发明技术能为利用健康人体内天然VEGFR1抗体发展全新的、低副作用的肿瘤免疫治疗方案奠定了现实基础。
发明内容
本发明的目的是提供一种检测VEGFR1自身抗体的线性抗原多肽，由此制备检测VEGFR1自身抗体的特异试剂，并设计了相应的实施优选操作流程方案。本发明共设计三种VEGFR1线性抗原多肽，其氨基酸序列见表1
表1.VEGFR1抗原多肽序列
众所周知，由于抗原抗体的结合实际上只发生在抗原决定簇与抗体结合位点之间，两者在空间结构和构型上越接近完全互补，则抗原抗体的结合就越稳定，特异性就越强，结合效率就越高。由于目标抗体及其结合位点结构是先决因素， 因此根据抗原决定簇即可决定整个蛋白抗原与抗体结合状态和亲和特性。
因此，本发明根据VEFGR1蛋白的生物学特性，针对多个表位进行免疫信息学预测和模拟，经分析与抗原性相关的各种参数，设计了上述三种在空间结构和构型上与目标抗体完全互补的线性多肽抗原的氨基酸序列。
大量的研究资料证实，VEGFR1是一种肿瘤相关抗原，在多种实体肿瘤中表达。故抗VEGFR1抗体可以认为是一种天然抗肿瘤抗体。在人体内执行免疫监视功能，防止肿瘤的形成与发展。本发明的技术正是填补了定量检测这种天然抗肿瘤抗体的空白，为血浆生物制品公司研发新产品和临床医学发展防治肿瘤的新措施提供了重要工具。例如，血浆生物制品公司可利用本发明技术筛选血浆，以制备富含VEGFR1抗体的丙种球蛋白，用于临床防治肿瘤；临床也可以直接给早期肿瘤病人局部治疗后(如手术或放疗后)输入利用本发明技术筛选的富含VEGFR1抗体的血浆，提高其免疫监视功能，预防肿瘤的复发和转移。这是本发明技术的应用价值所在。
表1中所列三种抗原多肽在ELISA法检测血浆VEFGR1自身抗体应用中应以高纯度制品实施，建议使用纯度不低于95％的制品。以下用本领域通用的“夹心对比检验模式”进行技术验证说明本发明可实现的技术效果。
本申请提供了一种利用表1中所列的三种抗原多肽检测血浆免疫标志物VEFGR1自身抗体的方法，其包括如下具体步骤：
1、操作前，每种抗原多肽用65％～70％乙酸溶解为5mg/ml储存液,然后等体积混合并放置-20℃(误差2℃以内)冰箱中保存；
2、操作开始时，首先将表1中所列三种抗原多肽的等比混合液用包被液稀释为10～50微克/ml，该包被液为0.1M磷酸盐缓冲液含0.15M氯化钠和10mM EDTA，酸碱度(pH)为7.0～7.4之间；
3、然后包被马来酰亚胺(Maleimide)活化的96孔检测板(Thermo Scientific,美国)，4℃过夜孵育后，用洗液洗板3遍,所述洗液为0.1M磷酸盐缓冲液含0.15M氯化钠和0.1％TWEEN-20，酸碱度(pH)为7.0～7.4之间；
4、然后分步加样分析如下：
a、待测血浆样本设双复孔，另设2个阴性对照孔(NC，参照物为不含血浆VEFGR1抗体的阴性对照液，藉此可获得表1所述的本发明三种抗原多肽在不含VEFGR1自身抗体的阴性环境下的指标值，目的是证明本发明组合物在不含VEFGR1自身抗体的情形下不会显示阳性结果)和2个阳性对照孔(PC，参照物为含VEFGR1抗体标准品的阳性对照液，藉此可获得表1所述的本发明三种抗原多肽在VEFGR1抗体标准品基准含量水平上的指标值，目的是证明本发明组合物在含有VEFGR1自身抗体的环境下能显示阳性结果，并可进一步作为基准值用于比较待测血浆样本指标值)；
b、用分析液将待测血浆样本1:200稀释，所述分析液与抗原包被液相同，即0.1M磷酸盐缓冲液含0.15M氯化钠和10mM EDTA，酸碱度(pH)为7.0～7.4之间，每孔加100μl,25℃孵育1-2小时，然后洗板3遍；
c、用所述分析液按照上述b步骤(即0.1M磷酸盐缓冲液含0.15M氯化钠和10mM EDTA，酸碱度为7.0～7.4之间)操作后，稀释辣根过氧化物酶标记的羊抗人IgG(用以验证血桨中被检测的物质是否是特异性抗体)，抗体工作范围为1:10000～1:50000，每孔加100μl，25℃孵育1-2小时；
d、以前述洗液(即0.1M磷酸盐缓冲液含0.15M氯化钠和0.1％TWEEN-20，酸碱度PH值为7.0～7.4之间)洗板3遍后，每孔加100μl 3,3',5,5'-四甲基联苯胺(TMB)及过氧化物酶混合液，室温避光20～30分钟；
e、每孔加50μl终止液10％硫酸溶液(10％H2SO4)，然后用酶标仪检测光密 度(OD)值，检测波长为450nm，参考波长为630nm。检测过程应在加入终止液后10分钟内完成，据此可定量分析个体血浆VEGFR1自身抗体水平。
5、在进行群体随机抽样分析时，可针对每一个体检测所得数据进行分析，采用阳性样品比值(Positive sample ratio，PSR)判定血浆中VEGFR1自身抗体的相对水平。PSR计算方法如下：PSR＝[VEGFR1OD值–NC OD值]/[PC OD值–NC OD值]。然后应用正态四分法(Q-Q)作图。
表1中所列三种多肽抗原多肽在实际应用中可制备成简单易用的便捷测试盒套装，可以非金属材料真空密封包装，4度(4℃)环境下可保存不低于六个月。因此，本申请还提供了一种包含表1中所列三种抗原多肽的测试盒。简言之，将三种抗原多肽混合液包被的马来酰亚胺(Maleimide)活化96孔检测板在45度(45℃)烤箱中干燥后，用非金属包装材料真空密封包装，由各一件试剂组合为一个便捷测试盒套装。建议三种抗原多肽均为纯度不低于95％的制品。
附图说明
图1血浆VEGFR1抗体浓度的四分位数分布图。图中横坐标为以PSR值表示的血浆VEGFR1抗体浓度，纵坐标为血浆VEGFR1抗体浓度四分位数分布范围，0点水平所对应的PSR值为中位数。
具体实施例
1.样本收集：收集121份正常成年人血浆样品，包括65例男性和56例女性。所述血样提供者均为经临床体检确定不存在已患有任何一类肿瘤的个体。操作前所有血浆样品均在负80度(-80℃)条件下保存，经核实保存时间未超过两年，没有反复冻融(次数不超过3次)的血浆样品。
2.样本检测：在4℃环境下解冻血浆样品，本实验所采用的表1中所列三种 抗原多肽由英国SEVERN BIOTECH有限公司合成，纯度为95％，具体进行如下操作步骤：
(1)操作前，每种抗原多肽用67％乙酸溶解为5.7mg/ml储存液,然后等体积混合并放置-20℃冰箱中保存；
(2)操作开始时，首先将三种抗原多肽混合液用包被液稀释为28.5微克/ml，该包被液为0.1M磷酸盐缓冲液含0.15M氯化钠和10mM EDTA，测得酸碱度(pH值)为7.2；
(3)然后包被马来酰亚胺(Maleimide)活化的96孔检测板(Thermo Scientific,美国)，4℃过夜16.5小时孵育后，用洗液洗板3遍,所述洗液为0.1M磷酸盐缓冲液含0.15M氯化钠和0.1％TWEEN-20，测得酸碱度pH值为7.3；
(4)然后分步加样分析如下：
a、待测血浆样本设双复孔，另设2个阴性对照孔(NC，参照物为Sigma-Aldrich公司提供的不含VEFGR1抗体的人血浆阴性对照液)和2个阳性对照孔(PC，参照物同样为Sigma-Aldrich公司提供的人血浆VEFGR1抗体标准品阳性对照液)；
b、用分析液将血浆1:200稀释，所述分析液与抗原包被液相同，为0.1M磷酸盐缓冲液含0.15M氯化钠和10mM EDTA，测得酸碱度(pH)为7.2，每孔加100μl，25℃孵育1.5小时；
c、以前述洗液(即0.1M磷酸盐缓冲液含0.15M氯化钠和0.1％TWEEN-20，测得酸碱度为7.2)洗板3遍后，用分析液稀释辣根过氧化物酶标记的羊抗人IgG(由Sigma-Aldrich公司提供)，抗体工作环境为1:28500，每孔加100μl，25℃孵育1.5小时；
d、以前述洗液(即0.1M磷酸盐缓冲液含0.15M氯化钠和0.1％TWEEN-20， 测酸碱度PH值为7.2之间)洗板3遍后，每孔加100μl 3,3',5,5'-四甲基联苯胺(TMB)及过氧化物酶混合液(由Life Technologies公司提供)，室温避光25分钟；
e、每孔加50μl终止液10％硫酸溶液(10％H2SO4，)，然后用酶标仪检测光密度(OD)值，检测波长为450nm，参考波长为630nm，加入终止液后10分钟内检测完毕。
3.结果分析：以下进一步依据实验结果针对实验对象人群进行VEGFR1自身抗体的分布对比分析。针对前述检测所得数据进行分析时，采用阳性样品比值(Positive sample ratio，PSR)判定血浆中VEGFR1自身抗体水平，PSR计算公式为：PSR＝[VEGFR1OD值–NC OD值]/[PC OD值–NC OD值]，并以正态四分法(Q-Q)作图获得下图。图中横坐标为以PSR值表示的血浆VEGFR1抗体浓度，纵坐标为血浆VEGFR1抗体浓度四分位数分布范围，0点水平所对应的PSR值为中位数。
如图1所示，血浆VEGFR1抗体浓度的中位数为PSR＝1.53，偏态系数S＝1.65，峰度系数K＝4.63，统计学检验呈高度显著(w＝0.89,p<0.0001)。以正态分布拟合曲线上向右侧拐点的最高值为阈值(cut-off＝3.8)，即在121份正常成年人血浆样品中有8份富含VEGFR1自身抗体，本实施例中检出该阈值以上的人数占比为6.6％。根据本实施例可知，随机抽取的正常成年人群的血浆VEGFR1抗体含量状况呈偏态分布，其中8例血浆样品属于颇具临床应用价值的富含天然VEGFR1自身抗体的宝贵血源。
本实施例中，在同步阴性对比试验证明本发明不存在误报结果的同时，上图横坐标体现的是以Sigma-Aldrich公司提供的人血浆VEFGR1抗体标准品阳性对照液为参照的每一个体血样PSR计算值，并据此标注得到上图。为了便于 理解，从另一个角度看，我们可将VEGFR1自身抗体浓度(PSR值)分布为4个区间，以下表显示本实施例中落入每个抗体浓度区间的人数比例。
附表.121份正常成年人血浆样品中VEGFR1自身抗体分布
在以上具体实施例中，我们采用国际权威生化试剂公司Sigma-Aldrich公司提供的人血浆VEFGR1抗体阴性对照液和阳性标准品对照液，通过夹心对比试验的结果证明：本发明设计的三种线性抗原多肽与血浆中VEFGR1自身抗体可发生特异性结合，即三种线性多肽抗原氨基酸序列在空间结构和构型上与VEFGR1抗体蛋白是有效互补的，由此可量化测定不同个体的血浆VEFGR1自身抗体水平。由此得到了一种实用的血浆VEFGR1自身抗体检测手段，同时由于本发明设计的三种线性多肽抗原制备时所使用的合成手段相对简单而成本相应适中，这就为下一步以富含天然VEFGR1自身抗体血浆治疗肿瘤的临床应用发展奠定了理论和实践基础。
Claims (6)
1.一种用于检测VEGFR1自身抗体的组合物，其包括如下3种抗原多肽:
H-DEGVYHCKATNQKGSVESSAYLTVQGTSDKSNLE-OH；
H-DLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSI-OH；
H-ESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAEL-OH。
2.根据权利要求1所述的组合物，其特征在于：组合物中各抗原多肽的比例为1:1:1。
3.一种检测个体中的VEGFR1自身抗体的方法，其包括将权利要求1中所述的3种抗原多肽等比混合，然后包被马来酰亚胺(Maleimide)活化的96孔检测板，4℃过夜孵育，洗板，再进行分步加样分析。
4.权利要求4所述的方法，其中分步加样分析包括将待测血浆样本设双复孔，同时设2个阴性对照孔和2个阳性对照孔，用分析液将血浆稀释，并稀释辣根过氧化物酶标记的羊抗人IgG，洗板，每孔加100μl3,3',5,5'-四甲基联苯胺(TMB)及过氧化物酶混合液，室温避光20～30分钟，每孔添加50μl终止液10％硫酸溶液(10％H2SO4)，然后用酶标仪检测光密度(OD)值，检测波长为450nm，参考波长为630nm。
5.权利要求1或2所述的组合物用于检测VEGFR1自身抗体的应用。
6.根据权利要求1或2所述组合物制成的测试盒套装，其特征在于：三种抗原多肽分别以非金属医用包装材料真空独立封装后，各取一件组合为一个测试盒套装备用。
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| JP2017522419A JP6423092B2 (ja) | 2014-11-04 | 2015-08-07 | 血漿免疫標識物−ｖｅｇｆｒ１自己抗体の検出に用いる抗原ポリペプチド及び応用 |
| EP15857030.9A EP3216460B1 (en) | 2014-11-04 | 2015-08-07 | Antigen polypeptide for detecting plasma immunological marker-vegfr1 autoantibody and use thereof |
| RU2017119400A RU2670052C1 (ru) | 2014-11-04 | 2015-08-07 | Антигенный полипептид для выявления в плазме крови иммунного маркера - аутоантитела к vegfr1 и его применение |
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| JP2017538920A (ja) | 2017-12-28 |
| EP3216460B1 (en) | 2019-11-20 |
| EP3216460A1 (en) | 2017-09-13 |
| KR20170082152A (ko) | 2017-07-13 |
| RU2670052C1 (ru) | 2018-10-17 |
| WO2016070662A1 (zh) | 2016-05-12 |
| JP6423092B2 (ja) | 2018-11-14 |
| US20180284116A1 (en) | 2018-10-04 |
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