CN115327134A - Erbb2 s310f突变的抗体在制备诊断检测胆囊癌中的应用 - Google Patents
Erbb2 s310f突变的抗体在制备诊断检测胆囊癌中的应用 Download PDFInfo
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Abstract
本发明提供了一种ERBB2S310F突变的抗体在制备诊断检测胆囊癌中的应用，该突变引起胆囊癌中PD‑L1表达升高，抗体特异性识别人的胆囊癌中ERBB2S310F突变，同时进行免疫组织化学检测；该抗体的制备包括：ERBB2氨基酸序列中第310位野生型的丝氨酸突变为苯丙氨酸，然后免疫兔子，经过纯化即可；之前对ERBB2S310F突变的检测主要通过外显子组测序，这样需要消耗的时间和费用较多，不利于临床开展对ERBB2S310F突变的检测。同时病理诊断较为便捷，也是肿瘤诊断的金标准，本发明依据ERBB2S310F突变多肽制备的抗体可以进行免疫组织化学的检测，并用于胆囊癌的临床病理诊断。
Description
技术领域
本发明属于抗体检测技术领域，具体涉及一种ERBB2 S310F突变的抗体在制备诊断检测胆囊癌中的应用。
背景技术
ERBB家族(epidermal growth factor receptor,ERBB,上皮生长因子家族)包括ERBB1/EGFR、ERBB2/HER2、ERBB3和ERBB4四个成员。其中ERBB2在包括乳腺癌、胆囊癌等多种肿瘤中存在突变，其中ERBB2 S310F突变是胆囊癌的显著突变。以往对ERBB2突变的检测主要以全外显子测序为主，在临床病理诊断中并不方便。
发明内容
针对现有检测中的不足，本发明的目的是提供一种ERBB2 S310F突变的抗体在制备诊断检测胆囊癌中的应用。
本课题组前期与上海仁济医院课题组合作通过对胆囊癌样本进行全外显子组测序，证实ERBB2 S310F突变是胆囊癌的显著突变，并且ERBB2突变在引起胆囊癌PD-L1表达升高，促进胆囊癌的免疫逃逸中发挥重要作用。而目前对ERBB2 S310F突变并没有特意的抗体进行检测。为此，根据免疫学抗原抗体的特异反应的原理及抗体制备过程，预制备特异识别ERBB2 S310F突变的抗体，用于临床病理诊断。
为达到上述目的，本发明的解决方案是：
第一方面，本发明提供了一种ERBB2 S310F突变的抗体在制备诊断检测胆囊癌中的应用。
进一步地，ERBB2 S310F突变引起胆囊癌中PD-L1表达升高。
进一步地，抗体特异性识别人的胆囊癌中ERBB2 S310F突变。
进一步地，抗体进行免疫组织化学检测。
第二方面，本发明提供了一种上述的ERBB2 S310F突变的抗体在制备检测胆囊癌试剂盒中的应用。该试剂盒还包括免疫组化检测相关试剂，包括免疫组化DAB显示液(50*)，DAB显示液稀释液和免疫组化用兔二抗。
第三方面，本发明提供了一种上述的ERBB2 S310F突变的抗体的制备方法，其包括：
ERBB2氨基酸序列(mRNA序列为NM_004448.3，氨基酸序列为NP_004439.2)中第310位野生型的丝氨酸(S)突变为苯丙氨酸(F)，然后免疫兔子，收集兔子血清经过纯化，得到ERBB2 S310F突变的抗体。
由于采用上述方案，本发明的有益效果是：
之前对ERBB2 S310F突变的检测主要通过外显子组测序，这样需要消耗的时间和费用较多，不利于临床开展对ERBB2 S310F突变的检测。同时病理诊断检测较为便捷，也是肿瘤诊断的金标准，本发明依据ERBB2 S310F突变多肽制备的抗体可以进行免疫组织化学的检测，并用于胆囊癌的临床病理检测。
附图说明
图1为本发明的一代测序检测胆囊癌样本中ERBB2基因图。
图2为本发明的免疫组化分析制备的抗体对野生型ERBB2和S310F突变的ERBB2的特异性图。
图3为本发明的抗体检测野生型ERBB2和S310F突变的ERBB2胆囊癌示意图。
具体实施方式
本发明提供了一种ERBB2 S310F突变的抗体在制备诊断检测胆囊癌中的应用。
为了能够更方便在临床中对胆囊癌ERBB2 S310F突变进行检测，用ERBB2 S310F的突变多肽(NYLSTDVGFCTLV)制备了能够特异识别ERBB2 S310F突变位点的兔多抗，可以方便用于胆囊癌病理检测。在大多数医院可通过病理检测胆囊癌或其他肿瘤中的ERBB2 S310F突变。
本发明制备的抗体使用的抗原是NYLSTDVGFCTLV这段多肽，是ERBB2氨基酸序列的302位到314位。其中这段多肽的第9位下划线F，即ERBB2氨基酸序列的310位，由野生型的丝氨酸(S)突变为苯丙氨酸(F)。使用这段多肽作为抗原免疫兔子后，收集血清经过纯化制备了特性识别ERBB2 S310F突变的抗体，并在胆囊癌临床样本中得到验证。
其中，该发明为兔多抗，主要成分为抗体，可以特异识别人的胆囊癌样本中ERBB2S310F突变，但并不识别野生型ERBB2，因此是具有特异性的。
具体地，对胆囊癌样本通过一代测序鉴定出ERBB2野生型和S310F突变型的样本(如图1)。然后对筛选的样本进行免疫组织化学染色，验证制备的抗体的特异性。图2和图3的免疫组化结果可见，在胆囊癌临床样本进行检测后，制备的特异识别S310F突变的抗体并不能与野生型的ERBB2发生反应，而对S310F突变的ERBB2具有明显的阳性检测结果，表明制备的抗体具有良好的特异性。
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做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。
实施例1：
(a)首先是ERBB2 S310F的突变多肽的制备:NYLSTDVGFCTLV-C，将第310位的丝氨酸(S)突变为苯丙氨酸(F)(此多肽片段的第九个氨基酸即为苯丙氨酸(F))，合成的多肽通过HPLC进行纯化，纯度>90％。
(b)其次是预采血，挑选健康的6周大小的新西兰大白兔两只(约2Kg)，将兔子固定脱毛，通过耳静脉抽取血10mL(血清5mL)，作为阴性参照用。
(c)然后是免疫兔子及取血，将合成的多肽用NaCl溶解制备抗原，免疫兔子。每只兔子初次免疫400μg抗原，约1mL抗原溶液，分别在背部、大腿根部通过肌肉注射完成。后续免疫每次可适当减少，免疫周期约20天，免疫4-5次。免疫兔子结束10天左右，进行测试取血。
(d)抗体效价检测，四组分别为：A预采血血清，B抗血清，C抗血清+野生型多肽，D抗血清+突变型多肽。将抗原预先加入96孔板，封闭，烘干后待用。将不同组的抗血清加入进行孵育，抗血清在原来的基础上倍比稀释，孵育兔二抗，加入显色底物，通过酶标仪进行检测，观察抗血清的作用。抗体效价检测达到1万以上后可对兔子进行最终取血。
(e)抗血清的纯化将抗血清用PBS稀释，通过平衡柱进行洗脱，-80℃保存。
(f)应用对纯化的抗血清，可进一步用于蛋白质免疫印迹、免疫组化等进行应用及验证。
实施例2：
ERBB2 S310F突变的抗体在临床诊断中的应用，主要通过对石蜡切片进行免疫组织化学染色，具体实验步骤如下：
(a)取胆囊癌石蜡切片，在60℃烘箱烘烤1h，使石蜡融化。
(b)在通风橱中，使切片依次浸泡经过二甲苯20min，二甲苯20min，100％乙醇5min，95％乙醇5min，80％乙醇5min，75％乙醇5min，50％乙醇5min。
(c)然后用自来水冲洗20min，加3％过氧化氢放置于60℃烘箱反应10min，PBS清洗3次，每次5min。
(d)用柠檬酸在高压条件下进行抗原修饰10min，然后取出放至室温。
(e)孵育制备的兔多抗(即ERBB2 S310F突变的抗体)，在4℃孵育过夜。
(f)PBS清洗3次，每次5min，室温孵育兔多抗20min。
(g)PBS清洗3次，每次5min，进行DAB显色，显微镜观察出现褐色沉淀即为阳性结果，可终止显色。
(h)加苏木素染色10s后，浸泡在自来水中清洗20min。
(i)然后在通风橱中，使切片依次浸泡经过50％乙醇5min，75％乙醇5min，95％乙醇5min，100％乙醇5min，二甲苯20min，二甲苯20min。
(j)中性树脂封片，显微镜观察染色结果。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例。本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (6)
1.一种ERBB2 S310F突变的抗体在制备诊断检测胆囊癌中的应用。
2.根据权利要求1所述的应用，其特征在于：所述ERBB2 S310F突变引起胆囊癌中PD-L1表达升高。
3.根据权利要求1所述的应用，其特征在于：所述抗体特异性识别人的胆囊癌中ERBB2S310F突变。
4.根据权利要求3所述的应用，其特征在于：所述抗体进行免疫组织化学检测。
5.一种如权利要求1所述的ERBB2 S310F突变的抗体在制备检测胆囊癌试剂盒中的应用。
6.一种如权利要求1所述的ERBB2 S310F突变的抗体的制备方法，其特征在于：其包括：ERBB2氨基酸序列中第310位野生型的丝氨酸(S)突变为苯丙氨酸(F)，然后免疫兔子，收集兔子血清经过纯化，得到ERBB2 S310F突变的抗体。
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