CN112345766B - 一种荧光-放射性联用的体外靶向筛选方法 - Google Patents
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Abstract
本发明提供了一种荧光‑放射性联用的体外靶向筛选方法,包括以下步骤:将待检测药物结合到固相载体上,加入放射性核素标记的目标分子和荧光探针标记的相同目标分子;形成竞争性结合后同步检测荧光和放射性信号,获得可相互验证的两组数据结果用于更准确的分析药物与目标分子的结合能力,从而实现靶向药物筛选。本发明方法可实现单次试验同时检测多个待测药物的结合荧光和放射性信号强度,迅速高效的筛选出对目标分子具有高特异性结合能力的药物。可以有效降低假阳性概率,具有高通量、高准确性、操作简单等特点。可以快速筛选出特异性结合目标分子的抗体药物,可开发为预靶标抗体用于放射性靶向治疗中扩展目标放射性药物的适应症。
Description
技术领域
本发明属于生物标记进行体外靶向筛选技术领域,具体涉及一种荧光-放射性联用的体外靶向筛选方法。
背景技术
目前常用针对靶向药物(如单抗或多肽等)的特异性结合体外靶向活性检测方法主要是基于化学发光或荧光等信号手段,利用抗原抗体结合进行免疫反应定性和定量分析,达到体外筛选出具有目标靶点靶向活性的药物目的。
放射性靶向药物研究中,是将化学发光或荧光信号替换为放射性核素标记所产生的放射性信号。然而各个方法均有一定优缺点,化学发光和荧光方法其灵敏性与放射性信号不一致,并且前两种方法需要在药物上进行发光基团或荧光基团修饰,而其修饰后的衍生物性质可能与放射性标记配体修饰的靶向药物不一致,因此其结果无法直接应用到放射性药物靶向性判断中。而放射性标记的筛选技术受限于核素性质与资源供给状态,对实验开展的硬件要求也较高,较难开展高通量的快速筛选研究。
基于上述技术现状,如何开发出一种能同时检测荧光信号,并且与放射性检验结果匹配度高的体外靶向筛选联合技术,能够实现更有效的在体外对放射性靶向药物进行快速筛选,减少后期研发成本,提高药物研发成功率,成为现阶段亟待解决的技术问题。
发明内容
本发明的目的就是为了解决现有技术存在的问题,而提供一种荧光-放射性联用的体外靶向筛选方法,本发明方法可以更有效的在体外对放射性靶向药物进行快速筛选,减少后期研发成本,提高药物研发成功率。
本发明的目的是提供一种荧光-放射性联用的体外靶向筛选方法,所述筛选方法包括以下步骤:
(1)将待检测药物结合到固相载体上,加入放射性核素标记的目标分子;
(2)向步骤(1)所得物中加入荧光探针标记的相同目标分子;
(3)对步骤(2)所得物进行同步检测荧光和放射性信号,获得可相互验证的两组数据结果用于更准确的分析药物与目标分子的结合能力,从而实现靶向药物筛选。
本发明提供的上述方法,同时利用了荧光及放射性核素来标记目标靶向分子,通过不同浓度梯度的放射性标记目标分子与等浓度的荧光修饰目标分子同时与待测药物的竞争结合,可实现单次试验同时检测多个待测药物的结合荧光和放射性信号强度,迅速高效的筛选出对目标分子具有高特异性结合能力的药物。该方法利用多信号数据自洽性可以有效降低假阳性概率,具有高通量、高准确性、操作简单等特点。利用该方法可以快速筛选出特异性结合目标分子的抗体药物,可开发为预靶标抗体用于放射性靶向治疗中扩展目标放射性药物的适应症。
进一步的是,步骤(1)中所述放射性核素标记的目标分子的制备方法为:将目标分子溶解于醋酸钠缓冲液中,使最终浓度为0.02-20μmol/ml;加入放射性核素0.5-10mCi,42-85℃金属浴中加热反应30-60min,即得放射性核素标记的目标靶向分子。
进一步的是,所述醋酸钠缓冲液的浓度为0.25M,pH为5.5;所述加入的放射性核素比活度为10-1000mCi/ml。
进一步的是,所述放射性核素标记的目标分子包括放射性核素或对应的稳定同位素标记的目标分子,其中,所述稳定同位素与目标分子的反应摩尔比为1:2-10。
进一步的是,所述放射性核素包括68Ga、177Lu、18F[AlF]或90Y;所述对应的稳定同位素包括69Ga、175Lu、19F[AlF]或89Y。
进一步的是,步骤(2)中所述荧光探针标记的目标分子的制备方法为:将步骤(1)获取的稳定同位素标记的目标分子溶于磷酸缓冲液(10mM,pH 7.4),使最终浓度为0.02-20μmol/ml,加入TCEP和马来酰亚胺荧光探针,室温反应2h,超滤或HPLC纯化,即得荧光探针标记的带有稳定同位素的目标靶向分子。
进一步的是,所述磷酸缓冲液的浓度为10mM,pH为7.4。
进一步的是,所述荧光探针采用FTSC或Cy5荧光物质进行标记,所述马来酰亚胺荧光探针包括Alexa Fluor 647maleimide、Cy5 maleimide或FITC-PEG-maleimide。
进一步的是,步骤(3)中所述同步检测荧光和放射性信号的方法为:将溶解于磷酸缓冲液(10mM,pH 7.4)的100μL待测药品(浓度2.5-10μg/ml)加入96孔白板中,4℃过夜孵育,每孔用200μL的含有1%的吐温-20的磷酸缓冲液(10mM,pH 7.4)清洗3次;每孔加入200μL的3%牛血清蛋白溶液(3g牛血清蛋白溶于100ml磷酸缓冲液(10mM,pH 7.4),37℃孵育2h;每孔加入25μL步骤(2)的荧光探针标记的目标分子及25μL步骤(1)的放射性核素标记的目标分子;其中荧光分子浓度恒定,放射性核素分子浓度为3倍梯度稀释的8个以上浓度;将白板37℃孵育1h,上清转移至透明96孔板后用酶标仪在根据荧光探针类型选择合适波长检测荧光强度,每孔白板用于全自动伽马免疫分析仪检测放射性CPM值,分别绘制荧光信号及放射性信号的结合曲线,从而实现双重验证筛选出对放射性目标分子具有结合能力的待测药品,并实现半定量分析。
进一步的是,所述标记的目标分子为生物分子,具体包括多肽或抗体。
本发明的有益效果如下:
本发明提供了一种荧光-放射性联用的体外靶向筛选方法,其适用于快速筛选出对目标分子具有免疫结合活性的多个药物。该方法利用多信号数据自洽性可以有效降低假阳性概率,具有高通量、高准确性、操作简单等特点。利用该方法可以快速筛选出特异性结合目标分子的抗体药物,可开发为预靶标抗体用于放射性靶向治疗中扩展目标放射性药物的适应症。
附图说明
图1是本发明所述的荧光-放射性联用体外筛选技术示意图;
图2是本发明实施例所述的荧光探针及放射性核素标记的目标分子衍生物的结构;
图3是本发明实施例所述的目标化合物荧光光谱强度-浓度标准曲线;
图4是本发明实例所述的放射性目标分子iTLC图谱;
图5是本发明实施例所述的针对目标分子的特异性结合单抗筛选数据结果;其中,左图为荧光结合结果,右图为放射性结合结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行具体描述,有必要指出的是,以下实施例仅仅用于对本发明进行解释和说明,并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整,仍属于本发明的保护范围。
实施例1
本实施例中,提供了一种放射性177Lu-DOTA-NGR多肽及其稳定同位素标记的175Lu-DOTA-NGR多肽的制备方法,具体步骤如下:
取2μl的DOTA-NGR溶于醋酸钠缓冲液(0.25M,pH 5.5)中的多肽溶液,加入20μl醋酸钠缓冲液(0.25M,pH 5.5)稀释至最终浓度为0.2μmol/ml;加入177Lu放射性核素1mCi(比活度为800mCi/ml),80℃金属浴中加热反应60min即得放射性核素标记的目标靶向分子177Lu-DOTA-NGR多肽。稳定同位素标记的目标分子制备步骤与上相同,只是将放射性核素177Lu替换为对应的稳定同位素175Lu,即得175Lu-DOTA-NGR多肽,稳定同位素与目标靶向分子反应比例为摩尔比1:10。通过HPLC对反应进行纯化,纯化后放化纯度>95%,化学纯度>95%。通过iTLC检测放射性标记率>99%。
实施例2
本实施例中,提供了一种荧光探针Alexa Fluor 647maleimide标记175Lu-DOTA-NGR多肽的制备方法,具体步骤如下:
取实施例1中的175Lu-DOTA-NGR多肽溶于20μl磷酸缓冲液(10mM,pH 7.4),使最终浓度为0.2μmol/ml,加入1.5μg的TCEP和2μg的马来酰亚胺荧光探针,室温反应4h,HPLC纯化分离即得荧光探针标记的带有稳定同位素的目标靶向分子,175Lu-DOTA-NGR-AlexaFluor647。
实施例3
本实施例中,提供了一种特异性结合177Lu-DOTA-NGR目标多肽分子的单抗药物的筛选方法,步骤如下:
取待测单抗药物10种分别溶解于磷酸缓冲液(10mM,pH 7.4)中,每种单抗药物终浓度均为5μg/ml,在96孔白板中加入每种抗体100μl,4℃过夜孵育,每孔用200μl的含有1%的吐温-20的磷酸缓冲液(10mM,pH 7.4)清洗3次;每孔加入200μl的3%牛血清蛋白溶液(3g牛血清蛋白溶于100ml磷酸缓冲液(10mM,pH 7.4)),37℃孵育2h;每孔加入25μl实施例2中的荧光探针标记的目标分子175Lu-DOTA-NGR-Alexa Fluor 647及25μl实施例1中的放射性核素标记的目标分子177Lu-DOTA-NGR;其中175Lu-DOTA-NGR-Alexa Fluor 647浓度为100ng/ml,177Lu-DOTA-NGR放射性用量从0-2μCi/孔,3倍梯度稀释;将白板37℃孵育1h,上清转移至透明96孔板后用酶标仪在根据荧光探针类型选择合适波长检测荧光强度,每孔白板用于全自动伽马免疫分析仪检测放射性CPM值;分别以177Lu-DOTA-NGR浓度梯度为横坐标,绘制荧光强度曲线及放射性计数曲线,其中荧光结合筛选出可特异性结合177Lu-DOTA-NGR的单抗药物2种,而放射性结合筛选出可特异性结合177Lu-DOTA-NGR单抗药物1种,综合判断抗体4的特异性结合能力最好,适用于进一步开发为靶向结合药物。
Claims (6)
1.一种荧光-放射性联用的体外靶向筛选方法,其特征在于,所述筛选方法包括以下步骤:
(1)将待检测药物结合到固相载体上,加入放射性核素标记的目标分子;所述放射性核素标记的目标分子包括放射性核素或对应的稳定同位素标记的目标分子,其中,所述稳定同位素与目标分子的反应摩尔比为1:2-10;所述放射性核素标记的目标分子的制备方法为:将目标分子溶解于醋酸钠缓冲液中,使最终浓度为0.02-20μmol/ml;加入放射性核素0.5-10mCi,42-85℃金属浴中加热反应30-60min,即得放射性核素标记的目标靶向分子;
(2)向步骤(1)所得物中加入荧光探针标记的相同目标分子;所述荧光探针标记的目标分子的制备方法为:将步骤(1)获取的稳定同位素标记的目标分子溶于浓度为10mM,pH为7.4的磷酸缓冲液,使最终浓度为0.02-20μmol/ml,加入TCEP和马来酰亚胺荧光探针,室温反应2h,超滤或HPLC纯化,即得荧光探针标记的带有稳定同位素的目标靶向分子;
(3)对步骤(2)所得物进行同步检测荧光和放射性信号,获得可相互验证的两组数据结果用于更准确的分析药物与目标分子的结合能力,从而实现靶向药物筛选。
2.根据权利要求1所述的荧光-放射性联用的体外靶向筛选方法,其特征在于,所述醋酸钠缓冲液的浓度为0.25M,pH为5.5;所述加入的放射性核素比活度为10-1000mCi/ml。
3.根据权利要求1所述的荧光-放射性联用的体外靶向筛选方法,其特征在于,所述放射性核素包括68Ga、177Lu、18F[AlF]或90Y;所述对应的稳定同位素包括69Ga、175Lu、19F[AlF]或89Y。
4.根据权利要求1所述的荧光-放射性联用的体外靶向筛选方法,其特征在于,所述荧光探针采用FTSC或Cy5荧光物质进行标记,所述马来酰亚胺荧光探针包括Alexa Fluor647maleimide、Cy5maleimide或FITC-PEG-maleimide。
5.根据权利要求1所述的荧光-放射性联用的体外靶向筛选方法,其特征在于,步骤(3)中所述同步检测荧光和放射性信号的方法为:将溶解于10mM,pH 7.4磷酸缓冲液的100μL待测药品加入96孔白板中,所述待测药品的浓度为2.5-10μg/ml,4℃过夜孵育,每孔用200μL的含有1%的吐温-20的10mM,pH 7.4磷酸缓冲液清洗3次;每孔加入200μL的3%牛血清蛋白溶液,所述3%牛血清蛋白溶液为3g牛血清蛋白溶于100ml浓度为10mM,pH为7.4的磷酸缓冲液,37℃孵育2h;每孔加入25μL步骤(2)的荧光探针标记的目标分子及25μL步骤(1)的放射性核素标记的目标分子;其中荧光分子浓度恒定,放射性核素分子浓度为3倍梯度稀释的8个以上浓度;将白板37℃孵育1h,上清转移至透明96孔板后用酶标仪在根据荧光探针类型选择合适波长检测荧光强度,每孔白板用于全自动伽马免疫分析仪检测放射性CPM值,分别绘制荧光信号及放射性信号的结合曲线,从而实现双重验证筛选出对放射性目标分子具有结合能力的待测药品,并实现半定量分析。
6.根据权利要求1所述的荧光-放射性联用的体外靶向筛选方法,其特征在于,所述标记的目标分子为生物分子,包括多肽或抗体。
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