CN106771200B - Igf2bp3和afp联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒 - Google Patents
Igf2bp3和afp联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒 Download PDFInfo
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Abstract
本发明公开了IGF2BP3和AFP联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒，涉及生物检测技术领域。本发明提供的IGF2BP3检测试剂盒，其包括用于特异性结合待检样本中的IGF2BP3的第一抗体，用于检测待检样本的IGF2BP3浓度，其中，IGF2BP3的氨基酸序列如SEQ ID NO.1所示，其配合现有的AFP的检测试剂盒，形成IGF2BP3‑AFP联合检测试剂盒，其中，AFP的氨基酸序列如SEQ ID NO.2所示，该IGF2BP3‑AFP联合检测试剂盒以IGF2BP3‑AFP作为联合标志物，为肝癌的诊断、疗效评估以及预后干预提供准确可靠的诊断结果。
Description
技术领域
本发明涉及生物检测领域，且特别涉及IGF2BP3和AFP联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒。
背景技术
肝细胞性癌(简称肝癌)是世界范围内发病率最高恶性肿瘤之一，每年新发病例约有100万。如何及早发现、及时干预、实现疗效评估和监测以及对术后患者进行实时的复发监控等是肝癌防治中亟待解决的问题。而灵敏度高、特异性强的肝癌诊断试剂的研制是提高肝癌早期检出率、改善患者预后的关键之一。
甲胎蛋白(α-fetoprotein，AFP)是肝癌细胞的敏感特异性标志物。正常成人血清AFP浓度的参考区间是1-20μg/L。一般检测血清AFP浓度大于500μg/L、且持续4周者或AFP在200-500μg/L、持续8周者，可考虑有患肝癌的可能；同时血清AFP水平还可用于肝癌疗效评估和预后判断。但在胃肠管肿瘤如胰腺癌、肺癌以及一些肝良性病变如肝炎、肝硬化中也会出现不同程度的AFP增高现象。因此，单独用血清AFP作为肝细胞癌的标志物存在一定程度的假阳性和假阴性。
发明内容
本发明的目的之一在于提供IGF2BP3检测试剂盒，其用于检测待检样本中的IGF2BP3浓度。本发明提供的IGF2BP3检测试剂盒稳定性良好，其对IGF2BP3浓度的检测，具有强的特异性和重复性。
本发明的目的之二在于提供IGF2BP3-AFP联合检测试剂盒，其用于检测待检样本中IGF2BP3-AFP的浓度。采用本发明提供的联合检测试剂盒，将IGF2BP3-AFP作为联合标志物对待检样本中的IGF2BP3和AFP的浓度分别进行检测，以此检测结果作为诊断依据，能有效地避免单独用AFP作为标志物进行肝癌诊断、疗效评估或预后干预判断中的假阳性和假阴性结果的出现。
本发明的目的之三在于提供IGF2BP3和AFP作为联合标志物在制备肝癌诊断试剂盒中的应用，在制备肝癌诊断试剂盒中，将IGF2BP3和AFP作为联合标志物，检测时，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现，为治疗肝癌诊断提供准确可靠的结果。
本发明的目的之四在于提供IGF2BP3和AFP作为联合标志物在制备肝癌疗效评估试剂盒中的应用，在制备肝癌疗效评估试剂盒中，将IGF2BP3和AFP作为联合标志物，检测时，能有效地避免单独用AFP作为标志物进行肝癌疗效评估中的假阳性和假阴性结果的出现，为治疗肝癌疗效评估提供准确可靠的结果。
本发明的目的之五在于提供IGF2BP3和AFP作为联合标志物在制备肝癌预后干预试剂盒中的应用，在制备肝癌预后干预试剂盒中，将IGF2BP3和AFP作为联合标志物，检测时，能有效地避免单独用AFP作为标志物进行肝癌预后干预判断中的假阳性和假阴性结果的出现，为肝癌预后干预判断提供准确可靠的结果。
本发明解决其技术问题是采用以下技术方案来实现的。
一种IGF2BP3检测试剂盒，其包括用于特异性结合待检样本中的IGF2BP3的第一抗体，IGF2BP3的氨基酸序列如SEQ ID NO.1所示。
一种IGF2BP3-AFP联合检测试剂盒，其包括上述的IGF2BP3检测试剂盒以及用于检测上述的待检样本中的AFP浓度的AFP检测试剂盒，AFP的氨基酸序列如SEQ ID NO.2所示。
IGF2BP3和AFP作为联合标志物在制备肝癌诊断试剂盒中的应用，IGF2BP3的氨基酸序列如SEQ ID NO.1所示，AFP的氨基酸序列如SEQ ID NO.2所示。
IGF2BP3和AFP作为联合标志物在制备肝癌疗效评估试剂盒中的应用，IGF2BP3的氨基酸序列如SEQ ID NO.1所示，AFP的氨基酸序列如SEQ ID NO.2所示。
IGF2BP3和AFP作为联合标志物在制备肝癌预后干预试剂盒中的应用，IGF2BP3的氨基酸序列如SEQ ID NO.1所示，AFP的氨基酸序列如SEQ ID NO.2所示。
本发明提供的IGF2BP3和AFP联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒的有益效果是：本发明首次发现，通过检测待检样本中的IGF2BP3和AFP浓度，并绘制IGF2BP3和AFP作为联合标志物的综合ROC曲线，与单独使用AFP作为标志物或单独使用IGF2BP3作为标志物绘制的ROC曲线相比，IGF2BP3和AFP作为联合标志物的ROC曲线的曲线下面积为0.822，灵敏度为62.8％，特异性为91.1％，其曲线下面积和特异性较单独使用AFP作为标志物或单独使用IGF2BP3作为标志物的曲线下面积和特异性有显著提高。由此说明，与以AFP单独作为标志物诊断肝癌相比，将IGF2BP3和AFP作为联合标志物诊断肝癌，具有更显著的诊断意义。并进一步说明，将IGF2BP3和AFP作为联合标志物在制备肝癌诊断试剂盒、肝癌疗效评估试剂盒、肝癌预后干预试剂盒中具有广阔前景和应用价值。以IGF2BP3和AFP作为联合标志物制备得到的肝癌诊断试剂盒、肝癌疗效评估试剂盒、肝癌预后干预试剂盒等用于检测或诊断肝癌时，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明实施例7中IGF2BP3检测试剂盒检测IGF2BP3的标准曲线；
图2为本发明实施例14中IGF2BP3-AFP联合检测试剂盒检测肝癌的ROC曲线。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本发明实施例的IGF2BP3和AFP联合标志物在制备肝癌诊疗评估试剂盒中的应用及试剂盒进行具体说明。
胰岛素样生长因子2信使RNA结合蛋白3(insulinolike Growth Factor 2 mRNA-Binding Protein 3，IGF2BP3)在RNA转位、固定、细胞生长及细胞迁移中扮演重要角色。正常情况下，IGF2BP3在人胚胎时期的肝、肾、肺和胸腺等组织中高度表达，而成人仅在扁桃体、淋巴细胞、卵巢、睾丸和胎盘中表达。近来研究发现：IGF2BP3在胰腺癌、肺癌、食管癌等多种恶性肿瘤组织中高度表达；在肝癌、肺癌、直肠癌病人的血液中高度表达。因此，IGF2BP3的高度表达是多种恶性肿瘤的一个特异性标志物，有助于肿瘤的筛查、早期诊断以及选择靶向治疗的靶标。
有鉴于此，首先，本发明提供了一种IGF2BP3检测试剂盒，其包括用于特异性结合待检样本中的IGF2BP3的第一抗体。其中，IGF2BP3的氨基酸序列如SEQ ID NO.1所示。该试剂盒稳定性良好，并且其对IGF2BP3的浓度检测具有较强的特异性和重复性。该IGF2BP3检测试剂盒可检测待检样本中的IGF2BP3的浓度，其可与检测同一待检样本中的AFP浓度的AFP检测试剂盒进行联合检测，根据检测出的IGF2BP3和AFP浓度作为联合标志物，为肝癌的诊断、疗效评估、预后干预等提供可靠准确的结果，其具有较好的灵敏度和特异性。
优选地，第一抗体选自鼠抗人IGF2BP3单克隆抗体、兔抗人IGF2BP3单克隆抗体、羊抗人IGF2BP3单克隆抗体或鸡抗人IGF2BP3单克隆抗体中的任意一种。
进一步地，本发明提供的IGF2BP3检测试剂盒中还包括用于结合第一抗体的第二抗体，第二抗体标记有用于催化显色底物显色的酶。优选地，酶为辣根过氧化物酶或碱性磷酸酯酶中的一种。第二抗体与第一抗体结合后，第二抗体标记的酶量就能间接反映出与第一抗体结合的IGF2BP3的量，以此间接测定待检样本中IGF2BP3的浓度。
优选地，第二抗体选自兔抗鼠抗人IGF2BP3单克隆抗体、兔抗鼠抗人IGF2BP3多克隆抗体、兔抗羊抗人IGF2BP3单克隆抗体、兔抗羊抗人IGF2BP3多克隆抗体、羊抗兔抗人IGF2BP3单克隆抗体、羊抗兔抗人IGF2BP3多克隆抗体、鼠抗鸡抗人IGF2BP3单克隆抗体或鼠抗鸡抗人IGF2BP3多克隆抗体中的任意一种。
优选地，本发明提供的IGF2BP3检测试剂盒中的第二抗体保存在稳定剂中。稳定剂包括体积分数为66％-70％的PBS，质量分数为28％～32％的BSA以及质量分数为0.018％～0.022％的NaN3。NaN3作为防腐剂，可抑制细菌生成以及蛋白的聚合，在稳定剂中添加0.018％～0.022％的NaN3可使第二抗体能够长期存储，使用时具有良好的生物学活性。
进一步地，本发明提供的IGF2BP3检测试剂盒还包括稀释液。优选地，稀释液按质量分数计包括0.8％～1.2％的BSA、0.04％～0.06％的Tween-20以及0.018％～0.022％的NaN3，溶剂为1×PBS。其中，Tween-20是一种非离子型表面活性剂，它不破坏蛋白结构，不与抗体交叉反应，同时具有复性抗原的作用，可提高第一抗体和第二抗体的特异性的识别能力；同样的，NaN3起防腐剂的作用，抑制细菌生成以及蛋白的聚合。
需要说明的是，本发明提供的IGF2BP3检测试剂盒还可以包括固相载体、IGF2BP3标准品、洗涤浓缩液、显色底物、终止液和封闭膜中的一种或多种。
固相载体用于包被第一抗体。固相载体可选自聚氯乙烯、聚苯乙烯、聚丙酰胺或纤维素中的任意一种；其形式可选自凹孔平板、试管或珠粒中的任意一种。需要说明的是，本发明提供的试剂盒中的第一抗体可以是以包被于固相载体的形式存在；也可以是以第一抗体和固相载体分开独立的形式存在，检测过程中，再将第一抗体包被于固相载体。
IGF2BP3标准品用于制作标准曲线。其状态可以是固体粉末，也可以是已知浓度的标准液。当其是固体粉末时，用稀释液配置成系列浓度梯度的工作液后使用；当其是已知浓度的标准液时，其优选地保存在稳定剂中，使用时，用稀释液稀释。
洗涤浓缩液为：包含有质量分数为4.5％的Tween-20的30×PBS缓冲液，经30倍稀释后使用。
显色底物应与第二抗体标记的酶对应。具体地，当酶为辣根过氧化物酶(HRP)时，显色底物包括显色液A和显色液B。其中，显色液A为过氧化氢，显色液B可以是四甲基联苯胺(TMP，检测波长为450nm)、5-氨基水杨酸(5-AS，检测波长为449nm)或邻苯二胺(OPD，检测波长为492nm)中的任意一种。当酶为碱性磷酸酯酶时，显色底物可以是萘酚-AS-Mx磷酸盐和重氮盐的组合物(检测波长为500nm)或4-硝基酚磷酸盐(PNP，检测波长为400nm)。
终止液为浓酸或浓碱。
封闭膜用于孵育时贴在固相载体上，防止孵育过程中液体的蒸发。
本发明提供的IGF2BP3检测试剂盒可以检测的待检样本选自血清、血浆、组织匀浆液、唾液、尿液或细胞培养上清液中的任意一种。待检样本应该清澈透明，悬浮物应离心去除。待检样本中IGF2BP3含量若高于IGF2BP3工作液的最高值，应做适当倍数的稀释。IGF2BP3标准液以及待检样本的稀释均选用稀释液作溶剂。
其次，本发明还提供了一种IGF2BP3-AFP联合检测试剂盒，其包括上述的任一项所述的IGF2BP3检测试剂盒以及用于检测同一待检样本中的AFP浓度的AFP检测试剂盒。其中，AFP的氨基酸序列如SEQ ID NO.2所示。
进一步地，本联合检测试剂盒中的AFP检测试剂盒可以是现有技术的任意一种AFP检测试剂盒，如AFP酶联免疫检测试剂盒，也可以是自行配制的用于检测AFP浓度的各种试剂的组合。
IGF2BP3-AFP联合检测试剂盒能够对待检样本的IGF2BP3和AFP浓度进行检测，以检测出的IGF2BP3和AFP作为联合标志物用于对肝癌诊断、疗效评估、预后干预等，其具有良好的灵敏度和特异性，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
再次，本发明提供了IGF2BP3和AFP作为联合标志物在制备肝癌诊断试剂盒中的应用。将IGF2BP3和AFP作为联合标志物检测肝癌，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
此外，本发明还提供了IGF2BP3和AFP作为联合标志物在制备肝癌疗效评估试剂盒中的应用。将IGF2BP3和AFP作为联合标志物进行肝癌疗效评估，能有效地避免单独用AFP作为标志物进行肝癌疗效评估中的假阳性和假阴性结果的出现。
再有，本发明还提供了IGF2BP3和AFP作为联合标志物在制备肝癌预后干预试剂盒中的应用。将IGF2BP3和AFP作为联合标志物进行肝癌预后干预判断，能有效地避免单独用AFP作为标志物进行肝癌预后干预判断中的假阳性和假阴性结果的出现。
利用IGF2BP3和AFP作为联合标志物，取代AFP单独作为标志物，运用于肝癌的诊断、疗效评估或预后干预，具体而言，就是通过分别检测待检样本中IGF2BP3和AFP的浓度，并通过统计学方法分别计算IGF2BP3和AFP的cutoff值，以评价IGF2BP3和AFP作为联合标志物相对于以IGF2BP3或AFP单独作为标志物，在肝癌的诊断、疗效评估或预后干预中的诊断价值。将IGF2BP3和AFP作为联合标志物检测肝癌，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
本发明将IGF2BP3和AFP作为联合标志物检测待检样本，其ROC曲线的曲线下面积为0.822，灵敏度为62.8％，特异性为91.1％。其曲线下面积和特异性较单独使用AFP作为标志物检测诊断肝癌时有显著提高。由此说明：将IGF2BP3和AFP作为联合标志物诊断肝癌，与以AFP单独作为标志物诊断肝癌相比，具有更显著的诊断意义。即将IGF2BP3和AFP作为联合标志物检测肝癌，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
综上，本发明提供的IGF2BP3检测试剂盒可用于检测待检样本中的IGF2BP3浓度，其配合现有的AFP的检测试剂盒，可制备IGF2BP3-AFP联合检测试剂盒。该联合检测试剂盒以IGF2BP3-AFP为联合标志物，分别检测出待检样本中的IGF2BP3和AFP浓度，为肝癌的诊断、疗效评估以及预后干预提供准确可靠的诊断结果。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例中提供的IGF2BP3检测试剂盒，其包括用于特异性结合IGF2BP3的第一抗体。该第一抗体为鼠抗人IGF2BP3单克隆抗体。其中，IGF2BP3的氨基酸序列如SEQ ID NO.1所示。
使用本实施中例中提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的方法如下：
S1：包被第一抗体。在酶标板(购自Greiner公司，货号705071)的各孔中同时加入50μL的pH9.6碳酸盐缓冲液以及100μL的5μg/mL鼠抗人IGF2BP3单克隆抗体，4℃条件下过夜包被，每孔300μL洗涤液洗涤3次，每次浸泡2分钟(以下简称洗板)。
S2：封闭多余间隙。加入100μL质量分数为2％的牛血清白蛋白(BSA)，25℃下封闭2小时，洗板。
S3：加入测试样本。分别设置对照孔、7个标准孔(用于制作标准曲线)和待检样本孔。对照孔中加入空白稀释液，7个标准孔中分别加入梯度浓度的IGF2BP3工作液(IGF2BP3工作液由IGF2BP3标准品(购自Novus Biological公司)用稀释液作溶剂配制而成，梯度浓度依次为0ng/L、32.5ng/L、75ng/L、150ng/L、300ng/L、600ng/L、1200ng/L)，待检样本孔中加入待检样本，加入的体积均为100μL。用封闭膜封闭酶标板，于37℃条件下孵育30分钟。
S4：加入酶标记的第二抗体。各孔中加入100μL浓度为0.5μg/mL的辣根过氧化物酶标记的兔抗鼠抗人IGF2BP3多克隆抗体(购自Santa Cruz公司)，在37℃条件下孵育30分钟，洗板。
S5：加入显色底物。各孔中加入100μL浓度为4g/L的四甲基联苯胺(TMB)以及10μL质量分数为0.02％的过氧化氢，在37℃条件下孵育15分钟。
S6：加入终止液。各孔中加入50μL浓度为2M的硫酸终止反应，并于450nm条件下检测各孔吸光度(OD值)。
以IGF2BP3工作液浓度为纵坐标，其OD值为横坐标，绘制标准曲线，并得到回归方程。将检测得到的待检样本的OD值代入回归方程，计算出待检样本中IGF2BP3的浓度。
需要说明的是，本实施例中所用的试剂未注明生产厂商者，均为可以通过市售购买获得的常规产品；所用方法未特别说明的，均采用常规方法。
实施例2
本实施例中提供的IGF2BP3检测试剂盒，与实施例1中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例中的IGF2BP3 检测试剂盒还包括用于结合第一抗体的酶标记的第二抗体。该第二抗体为兔抗鼠抗人IGF2BP3多克隆抗体，酶为辣根过氧化物酶。第二抗体保存于稳定剂中，稳定剂包括体积分数为68％的PBS，质量分数为30％的BSA以及质量分数为0.02％的NaN3。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例1。
实施例3
本实施例中提供的IGF2BP3检测试剂盒，与实施例2中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例中的稳定剂包括体积分数为66％的PBS，质量分数为28％的BSA以及质量分数为0.018％的NaN3。除此之外，本实施例中的IGF2BP3检测试剂盒还包括固相载体和封闭膜，该固相载体为96孔酶标板(12条×8孔)。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例1。
实施例4
本实施例中提供的IGF2BP3检测试剂盒，与实施例3中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例中的IGF2BP3检测试剂盒还包括显色底物和终止液，该显色底物包括显色液A和显色液B，其中显色液A为质量分数为0.02％的过氧化氢，显色液B为4g/L的四甲基联苯胺；终止液为浓度为2M的硫酸。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例1。
实施例5
本实施例中提供的IGF2BP3检测试剂盒，与实施例4中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例中的稳定剂包括体积分数为70％的PBS，质量分数为32％的BSA以及质量分数为0.022％的NaN3。除此之外，本实施例中的IGF2BP3检测试剂盒还包括IGF2BP3标准品。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例1。
实施例6
本实施例中提供的IGF2BP3检测试剂盒，与实施例5中提供的试剂盒大致相同，不同的是，本实施例中的IGF2BP3检测试剂盒还包括稀释液，稀释液按质量分数计包括0.8％的BSA、0.04％的Tween-20以及0.018％的NaN3，溶剂为1×PBS。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例1。
实施例7
本实施例中提供的IGF2BP3检测试剂盒，其包括：
酶标板：已包被鼠抗人IGF2BP3单克隆抗体的96孔酶标板；
酶标记的第二抗体：0.5μg/mL辣根过氧化物酶标记的兔抗鼠抗人IGF2BP3多克隆抗体；
标准液：用稳定剂(包括体积分数为68％的PBS，质量分数为30％的BSA以及质量分数为0.2％的NaN3)配制而成的2400ng/L的IGF2BP3标准液；
稀释液：按质量分数计包括1％的BSA、0.05％的Tween-20以及0.02％的NaN3，溶剂为1×PBS；
洗涤浓缩液：包含有质量分数为4.5％的Tween-20的30×PBS缓冲液；
显色底物：显色液A，质量分数为0.02％的过氧化氢；显色液B，浓度为4g/L的四甲基联苯胺；
终止液：浓度为2M的硫酸；
封闭膜。
运用本实施例中提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的方法如下：
S1：加入测试样本。分别设置对照孔、7个标准孔(用于制作标准曲线)和待检样本孔。对照孔中加入空白稀释液，7个标准孔中分别加入梯度浓度的IGF2BP3工作液(由稀释液梯度稀释而成，梯度浓度依次为0ng/L、32.5ng/L、75ng/L、150ng/L、300ng/L、600ng/L、1200ng/L)，待检样本孔中加入待检样本，加入的体积均为100μL。用封闭膜封闭酶标板，于37℃条件下孵育30分钟。
S2：加入酶标记的第二抗体。各孔中加入100μL浓度为0.5μg/mL的辣根过氧化物酶标记的兔抗鼠抗人IGF2BP3多克隆抗体，在37℃条件下孵育30分钟，洗板。
S3：加入显色底物。各孔中加入100μL浓度为4g/L的四甲基联苯胺(TMB)以及10μL质量分数为0.02％的过氧化氢，在37℃条件下孵育15分钟。
S4：加入终止液。各孔中加入50μL浓度为2M的硫酸终止反应，并于450nm条件下检测各孔吸光度(OD值)。
以IGF2BP3工作液浓度为纵坐标，其OD值为横坐标，绘制标准曲线，并得到回归方程。将检测得到的待检样本的OD值代入回归方程，计算出待检样本中IGF2BP3的浓度。
根据上述方法绘制的标准曲线如图1所示，IGF2BP3线性范围为75ng/L-1200ng/L，在线性范围内IGF2BP3工作液线性相关系数R2为0.9951，回收率在90％～110％范围内。
实施例8
本实施例中提供的IGF2BP3检测试剂盒，与实施例7中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例的IGF2BP3检测试剂盒中：第一抗体为羊抗人IGF2BP3单克隆抗体；酶标记的第二抗体为：辣根过氧化物酶标记的兔抗羊抗人IGF2BP3多克隆抗体；显色液B为邻苯二胺。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例7。
实施例9
本实施例中提供的IGF2BP3检测试剂盒，与实施例7中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例的IGF2BP3检测试剂盒中：第一抗体为兔抗人IGF2BP3单克隆抗体；酶标记的第二抗体为：辣根过氧化物酶标记的羊抗兔抗人IGF2BP3多克隆抗体；显色液B为5-氨基水杨酸。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例7。
实施例10
本实施例中提供的IGF2BP3检测试剂盒，与实施例7中提供的IGF2BP3检测试剂盒大致相同，不同的是，本实施例的IGF2BP3检测试剂盒中：稀释液按质量分数计包括1.2％的BSA、0.06％的Tween-20以及0.022％的NaN3，溶剂为1×PBS；第一抗体为鸡抗人IGF2BP3单克隆抗体；酶标记的第二抗体为：碱性磷酸酯酶标记的鼠抗鸡抗人IGF2BP3多克隆抗体；显色底物为4-硝基酚磷酸盐。
使用本实施提供的IGF2BP3检测试剂盒检测待检样本中IGF2BP3浓度的检测方法同实施例7。
实施例11
对实施例7提供的IGF2BP3检测试剂盒进行重复性和稳定性验证实验。
根据实施例7中的绘制的标准曲线以及检测方法，用实施例7提供的同一IGF2BP3检测试剂盒检测已知浓度(分别为1200ng/L和600ng/L)的IGF2BP3标准液。分别重复检测10次，检测结果显示，组内变异系数CV≤10％。用实施例7中提供的3个批号的IGF2BP3检测试剂盒检测同一待检样本，结果显示，3个批号IGF2BP3检测试剂盒的批间变异系数CV≤15％。由此说明，实施例7中提供的IGF2BP3检测试剂盒检测IGF2BP3浓度的重复性强。
将实施例7提供的IGF2BP3检测试剂盒在4℃条件下保存10个月、开封后在4℃条件下保存2个月或在0-4℃运输7天，各组份均能保持稳定。由此说明，实施例7中提供IGF2BP3检测的试剂盒稳定性良好。
实施例12
本实施例中提供的IGF2BP3-AFP联合检测试剂盒，包括实施例1至10中任一项的IGF2BP3的检测试剂盒以及用于检测AFP的AFP酶联免疫检测试剂盒。其中，AFP的氨基酸序列如SEQ ID NO.2所示。
本实施例提供的IGF2BP3-AFP联合检测试剂盒可以分别针对待检样本的IGF2BP3和AFP的浓度检测，以检测出的IGF2BP3和AFP作为联合标志物用于对肝癌诊断、疗效评估、预后干预等，其具有良好的灵敏度和特异性，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现。
实施例13
本实施例中提供的IGF2BP3-AFP联合检测试剂盒，包括实施例7中提供的IGF2BP3检测试剂盒以及AFP酶联免疫检测试剂盒(购自上海酶联生物科技有限公司，货号：GR-E10784)。
使用本实施例中提供的联合检测试剂盒检测待检样本中IGF2BP3-AFP联合标志物浓度的方法如下：
检测待检样本中的IGF2BP3浓度：
S1：加入测试样本。分别设置对照孔、7个标准孔(用于制作标准曲线)和待检样本孔。对照孔中加入空白稀释液，7个标准孔中分别加入梯度浓度的IGF2BP3工作液(由稀释液梯度稀释而成，梯度浓度依次为0ng/L、32.5ng/L、75ng/L、150ng/L、300ng/L、600ng/L、1200ng/L)，待检样本孔中加入待检样本，加入的体积均为100μL。用封闭膜封闭酶标板，于37℃条件下孵育30分钟。
S2：加入酶标记的第二抗体。各孔中加入100μL浓度为0.5μg/mL的辣根过氧化物酶标记的兔抗鼠抗人IGF2BP3多克隆抗体，在37℃条件下孵育30分钟，洗板。
S3：加入显色底物。各孔中加入100μL浓度为4g/L的四甲基联苯胺(TMB)以及10μL质量分数为0.02％的过氧化氢，在37℃条件下孵育15分钟。
S4：加入终止液。各孔中加入50μL浓度为2M的硫酸终止反应，并于450nm条件下检测各孔吸光度(OD值)。
以IGF2BP3工作液浓度为纵坐标，其OD值为横坐标，绘制标准曲线，并得到回归方程。将检测得到的待检样本的OD值代入回归方程，计算出待检样本中IGF2BP3的浓度。
检测待检样本中的AFP浓度的具体方法参见AFP酶联免疫检测试剂盒(购自上海酶联生物科技有限公司，货号：GR-E10784)的说明书。
本实施例提供的IGF2BP3-AFP联合检测试剂盒的效果同实施例13。
实施例14
IGF2BP3-AFP联合检测试剂盒的灵敏度和特异性验证。
从上海东方肝胆外科医院收集78例肝癌患者术前血清，同时收集79例健康人员(含肝硬化和无肝硬化)血清，每例血清0.5mL。然后采用实施例13提供的联合检测试剂盒及检测方法检测肝癌患者和健康人员血清中IGF2BP3和AFP标志物的浓度。
采用统计软件SPSS 2.0分别统计IGF2BP3和AFP标志物区分肝癌和正常人的cutoff值(阳性和阴性结果的临界值)。结果显示，AFP区分健康人和肝癌患者的cutoff值为30μg/L，IGF2BP3区分健康人和肝癌患者的cutoff值为985.4ng/L。
同时根据检测结果绘制ROC曲线。在本实施例中，ROC曲线是以不同IGF2BP3或AFP浓度值为阳性标准对已知血清进行检测，根据金标准，评价单独以IGF2BP3或AFP为标志物或以IGF2BP3和AFP为联合标志物诊断时的灵敏度和特异度，然后以1-特异度为横坐标，以灵敏度为纵坐标绘图，以判断该单独以IGF2BP3或AFP为标志物，或以IGF2BP3和AFP为联合标志物时，检测体系的诊断价值。
ROC曲线即接受者操作特性曲线，又称为感受性曲线(sensitivity curve)。曲线上各点反映着相同的感受性，它们都是对同一检测结果的反应，是在几种不同的判定标准下所得的结果。ROC曲线是根据一系列不同的二分类方式(分界值或决定阈)，以真阳性率(灵敏度)为纵坐标，假阳性率(1-特异度)为横坐标绘制的曲线。通常用曲线下面积(AUC)评价诊断价值，AUC越大越具有诊断价值：一般认为AUC在0.5至0.7之间具有较低的诊断价值，在0.7至0.9之间具有中等的诊断价值，大于0.9具有很高的诊断价值。
检测结果如表1和图2所示。图2中：a为单独以AFP为标志物检测的曲线，b为单独以IGF2BP3为标志物检测的曲线，c为以AFP-IGF2BP3作为联合标志物检测的曲线，d为参考线。
具体地：单独以AFP为标志物检测的曲线下面积为0.674，灵敏度为67.9％，特异性为67.1％；单独以IGF2BP3为标志物的曲线下面积为0.777，灵敏度为69.2％，特异性为82.3％。将IGF2BP3和AFP作为联合标志物时曲线下面积为0.822，灵敏度为62.8％，特异性为91.1％。由此可知，使用IGF2BP3和AFP作为联合标志物检测肝癌的曲线下面积及特异性显著优于单独使用IGF2BP3或AFP单一标志物检测肝癌。
表1.IGF2BP3-AFP作为联合标志物检测诊断肝癌的结果
| 标志物 | 曲线下面积 | 灵敏度(％) | 特异性(％) |
| IGF2BP3 | 0.777 | 69.2 | 82.3 |
| AFP | 0.674 | 67.9 | 67.1 |
| IGF2BP3-AFP | 0.822 | 62.8 | 91.1 |
根据检测结果，在假阳性率为20％条件下，应用Logistic回归统计方法计算得到IGF2BP3和AFP作为联合标志物诊断肝癌的判断公式，具体为：P＝exp(-3.209+0.003X1+0.004X2)/[1+exp(-3.209+0.003X1+0.004X2)](X1为IGF2BP3的实际检测值(ng/L)；X2为AFP的实际检测值(μg/L))；当P＞0.455时，判断为阳性；当P≤0.455时，判断为阴性。
实施例15
本发明的IGF2BP3-AFP联合检测试剂盒用于肝癌检测。
利用本发明的IGF2BP3-AFP联合检测试剂盒对50例疑似肝癌患者进行检测，然后根据P＝exp(-3.209+0.003X1+0.004X2)/[1+exp(-3.209+0.003X1+0.004X2)](X1为IGF2BP3的实际检测值(ng/L)；X2为AFP的实际检测值(μg/L))计算P值。结果显示，50例被检样本中，37例P值大于0.455，其中31例B超确认为肝癌患者；13例P值小于0.455，其中2例B超确认为肝癌患者，其余11例为其他良性肿瘤非肝癌患者，总体准确率为84％，假阴性率为4％，假阳性率为12％，总体诊断效果良好。
实施例16
本发明的IGF2BP3-AFP联合检测试剂盒用于肝癌预后干预。
对3例经过手术及相应后续疗程结束后的肝癌患者进行跟踪随访(患者来自上海东方肝胆外科医院)，治疗后三个月首次采集患者血清，采用本发明的IGF2BP3-AFP联合检测试剂盒检测患者血清中IGF2BP3和AFP浓度，以后每隔3-6个月检测一次，跟踪检测二十四个月，共检测5次。然后据联合检测判断公式P＝exp(-3.209+0.003X1+0.004X2)/[1+exp(-3.209+0.003X1+0.004X2)](X1为IGF2BP3的实际检测值(ng/L)；X2为AFP的实际检测值(μg/L))计算P值，当P＞0.455时，说明患者已发生转移复发；当P≤0.455时，说明患者未发生转移复发，即无进展生存。12个月后，医生根据临床症状判断肝癌患者是否发生转移复发，结果如表2所示。
表2.IGF2BP3-AFP联合检测试剂盒评估肝癌疗效结果
| 患者编号 | 3个月 | 6个月 | 12个月 | 18个月 | 24个月 | 临床评价 |
| 1 | P＝0.25 | P＝0.16 | P＝0.32 | P＝0.21 | P＝0.35 | 无进展生存 |
| 2 | P＝0.36 | P＝0.41 | P＝0.77 | -- | -- | 转移复发 |
| 3 | P＝0.17 | P＝0.24 | P＝0.22 | P＝0.27 | P＝0.32 | 无进展生存 |
由表2可知，检测结果是3例患者中有1例发生了肝癌转移复发，其余2例未发生转移复发，为无进展生存。因此，使用IGF2BP3-AFP联合检测试剂盒监控肝癌预后，可提示肝癌患者复发转移，为医生提前进行预后干预提供指导。
综上所述，本发明中提供的IGF2BP3检测试剂盒具有良好的稳定性，其对IGF2BP3的浓度检测具有强的特异性和重复性，可用于制备肝癌诊断、疗效评估或预后干预试剂盒。进一步将其联合现有的AFP检测试剂盒，用于制备IGF2BP3-AFP联合检测试剂盒，并用该联合试剂盒检测待检样本，根据检测结果绘制ROC曲线，以评价其在肝癌诊断中的诊断价值，结果显示：检测联合标志物，曲线下面积为0.822，灵敏度为62.8％，特异性为91.1％。其曲线下面积和特异性较单独使用AFP作为标志物检测时有显著提高。将本发明提供的IGF2BP3-AFP联合检测试剂盒用于肝癌疑似病例检测，总体准确率为84％；将其用于预后干预判断，可为医生提前进行预后干预提供可靠的指导。由此说明：将IGF2BP3和AFP联合作为标志物诊断肝癌、评估肝癌疗效以及判断肝癌预后干预效果与以AFP单独作为标志物相比，能有效地避免单独用AFP作为标志物诊断肝癌的假阳性和假阴性结果的出现，具有更科学的诊断意义。
以上所描述的实施例是本发明一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
序列表
<110> 上海缔达生物科技有限公司
<120> IGF2BP3和AFP作为联合标志物在制备肝癌诊断、疗效评估或预后干预试剂盒中的应用以及试剂盒
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Claims (3)
1.IGF2BP3和AFP作为联合标志物在制备肝癌诊断试剂盒中的应用，其特征在于，所述IGF2BP3的氨基酸序列如SEQ ID NO.1所示，所述AFP的氨基酸序列如SEQ ID NO.2所示。
2.IGF2BP3和AFP作为联合标志物在制备肝癌疗效评估试剂盒中的应用，其特征在于，所述IGF2BP3的氨基酸序列如SEQ ID NO.1所示，所述AFP的氨基酸序列如SEQ ID NO.2所示。
3.IGF2BP3和AFP作为联合标志物在制备肝癌预后干预试剂盒中的应用，其特征在于，所述IGF2BP3的氨基酸序列如SEQ ID NO.1所示，所述AFP的氨基酸序列如SEQ ID NO.2所示。
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