CN116735864B - 评估TfR1抗体血液安全性的试剂盒 - Google Patents
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Abstract
本发明适用于分子生物学技术领域,提供了一种用于进行TfR1抗体血液安全性评估的试剂盒,包括试剂R1和试剂X。其中,所述试剂R1为标记的Human Transferrin protein抗原;所述试剂X为R2或R3,其中,所述试剂R2为AF2474,所述R3为不和Tf识别相同抗原结合位点的TfR1抗体。所述试剂盒还包含样品稀释液、底物显色液、终止液和/或浓缩洗涤液。本发明还提供了进行TfR1抗体血液安全性评估的方法,所述方法包括:1)将试剂X包被于酶标板中;2)将试剂R1加入酶标板;3)按照梯度稀释依次加入待测抗体;4)加入HRP标记的SA;5)加入快速显色指示剂;6)测量吸光度OD值,绘制曲线,进行评估。本发明提供的试剂盒能灵敏、准确地评价TfR1抗体血液安全性。
Description
技术领域
本发明属于分子生物学技术领域,涉及一种评估TfR1抗体血液安全性的试剂盒及筛选TfR1抗体的方法。
背景技术
转铁蛋白又名运铁蛋白(Transferrin,Tf)是血浆中主要的含铁蛋白质,负责运载由消化管吸收的铁和由红细胞降解释放的铁,其以三价铁复合物(TRF-Fe³)的形式进入骨髓中,供成熟红细胞的生成。转铁蛋白受体(Transferrin receptor,TfR)是机体介导铁代谢的重要蛋白质分子,在铁的运输、转化和利用中起着关键作用。
转铁蛋白Tf结合铁是通过与其受体TfR1相互作用而实现的。Tf与Fe3+相互作用形成全铁-Tf,并与TfR1受体结合,在细胞内吞作用下进入核内体。在偏酸性核内体的环境中,Fe3+与Tf分离,同时STEAP3将Fe3+还原为Fe2+,并被二价金属离子转运蛋白1(DivalentMetal Transporter 1,DMT1)转运到细胞质中,然后释放了Fe3+的Tf与TfR1组成Tf/TfR1复合物,通过胞吐作用回游到细胞表面。在细胞表面,转铁蛋白Tf与受体TfR1分离,成为脱铁-Tf,然后再与Fe3+重新结合参与铁循环,整个过程完成后Tf和TfR1被循环利用,进入细胞摄取铁的下一个周期中。
目前已知两种不同类型的转铁蛋白受体即转铁蛋白受体1(TfR1)和转铁蛋白受体2(TfR2)。转铁蛋白受体是由两个大小约为90kDa的亚单位通过两条二硫键交联而成的一种Ⅱ型跨膜糖蛋白。每个单体含760个氨基酸,分子量为90~95kDa,包含一个大的胞外C端区域(671个氨基酸)、一个单跨膜区域(28个氨基酸)及一个短的N端区域(61个氨基酸)。C端区域是一个外功能区,它包含转铁蛋白(Tf)的结合位点、3个N连接糖基化位点及一个O连接糖基化位点,这些位点的糖基化作用是TfR1功能所必需的。
TfR在正常组织细胞中表达较低,而在肿瘤细胞表面表达增加,由于肿瘤细胞增长快速,需要合成更多DNA,核苷酸还原酶需要更多的辅因子铁,从而迫使肿瘤细胞上调TfR的表达。文献报道证明,肿瘤细胞表面的TfR与转铁蛋白的亲和力是正常细胞的10~100倍。同时,TfR的表达水平与肿瘤的分期和预后息息相关。TfR在肿瘤细胞高表达,相对特异性强,其胞外段捕捉配体转铁蛋白(transferrin, Tf)后介导内吞。这些特征使TfR成为抗肿瘤靶向治疗中的热点靶点之一。目前已设计有各种TfR1靶向抗体,被试图用于抑制肿瘤细胞生长,大多数TfR1抗体要么起运输作用,要么直接抑制受体功能。
但当TfR1抗体与TfR1结合时,如果侵占了TfR1和Tf复合体上Tf与Fe3+的结合位点,会使得其转铁功能受阻,进而影响人体健康。因此评估TfR1抗体的血液安全性显得尤为重要。而目前市面上并没有能够快速且准确进行TfR1抗体血液安全性评估的试剂盒及方法,因此,有必要设计一种临床前快速且准确进行TfR1抗体血液安全性评估试剂盒及方法。
发明内容
本发明实施例的目的在于提供一种临床前快速且准确进行TfR1抗体血液安全性评估的试剂盒,该试剂盒包括试剂R1和试剂X。
具体地,所述试剂R1为用Biotin标记的Human Transferrin R/CD71抗原,其在酶标板上的包被浓度为0.05~1 μg/mL。
具体地,所述试剂X为试剂R2或试剂R3。
具体地,所述试剂R2优选为AF2474抗体,其结合Human Transferrin R/CD71抗原,且同时和Transferrin(Tf)识别相同抗原结合位点。
具体地,所述试剂R3是和Human Transferrin R/CD71抗原结合的抗体,同时不和Transferrin(Tf)识别相同抗原结合位点。
进一步地,所述试剂盒还包含样品稀释液、底物显色液、终止液和/或浓缩洗涤液。
具体地,所述样品稀释液为CBS(pH9.2~9.6的碳酸盐缓冲液)。
具体地,所述底物显色液为双组分TMB显色液。
具体地,所述终止液为1 M盐酸溶液。
具体地,所述浓缩洗涤液为含0.05%吐温20的pH 7.4的磷酸盐缓冲液。
本发明实施例的目的还在于提供一种筛选TfR1抗体的方法,该方法筛选的抗体具有血液安全性,该方法包括:
1)将试剂X包被于酶标板中;
2)将试剂R1加入封闭后的酶标板;
3)按照4倍梯度稀释依次加入待测抗体;
4)加入HRP标记的链霉亲和素磁珠(SA);
5)加入快速显色指示剂;
6)测量吸光度OD值,并绘制拟合曲线,根据结果对抗体进行筛选。
具体地,所述待测样品为TfR1抗体,可以是单克隆抗体、双特异性抗体、多特异性抗体、或抗体片段,可以是具有特定组织靶向性的长度不少于10个氨基酸的蛋白质,可以是环状或线性的多肽。
与现有技术相比,本发明具有以下优点:
1)本发明提供的试剂盒具有检测灵敏度高、特异性强、检测重复性高和稳定性好等优点,能灵敏、准确地评价TfR1抗体血液安全性,可用于提供准确的治疗药物监测数据,实现TfR1药物的精准治疗。
2)本发明提供的方法能够快速且准确地顺利筛选出不影响转铁蛋白受体1功能的抗体,并能筛选出与TfR1的亲和力高于R3的抗体。
附图说明
图1是本发明实施例提供的验证R2阻断Tf和TfR1结合实验1的IC50曲线图。
图2是本发明实施例提供的验证R2阻断Tf和TfR1结合实验2的IC50曲线图。
图3是本发明实施例提供的用本发明的评估TfR1抗体血液安全性试剂盒对待测抗体安全性进行评估的结果曲线图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明实施例提供一种临床前快速且准确进行TfR1抗体血液安全性评估的试剂盒,该试剂盒包括试剂R1和试剂X。
具体地,所述R1为用Biotin标记的Human Transferrin R/CD71抗原,可以为重组表达的抗原,也可以为纯化的天然来源的抗原,其在酶标板上的包被浓度为0.05~1 μg/mL。
具体地,所述试剂R2是AF2474抗体,其和Human Transferrin R/CD71抗原结合,并且与Transferrin(Tf)识别相同抗原结合位点,两者形成竞争性结合关系。
具体地,所述试剂R3是和Human Transferrin R/CD71抗原结合的抗体,同时不和Transferrin(Tf)识别相同抗原结合位点。
进一步地,所述试剂盒还包含样品稀释液、底物显色液、终止液和/或浓缩洗涤液。
具体地,所述样品稀释液为含0.0189 M Na2CO3、0.0277 M NaHCO3的pH为9.6的碳酸盐缓冲液。
具体地,所述底物显色液为双组分TMB显色液。
具体地,所述终止液为1 M 盐酸。
具体地,所述浓缩洗涤液为含0.05%吐温20的pH7.4的磷酸盐缓冲液,在使用前稀释。
本发明实施例还提供一种对TfR1抗体进行筛选的方法,经筛选后的抗体具有高的血液安全性,能用于临床应用,该方法包括:
1)将1 μg/ml的试剂X(CBS,100 ul/well)于4℃包被于酶标板中过夜孵育,经0.05% PBST缓冲液洗涤后,经3% MPBS缓冲液于37℃封闭1h,再经0.05% PBST冲洗;
2)将30 μg/ml的试剂R1(50 μl/well)加入封闭后的酶标板;
3)加入待测抗体15 μg/ml,按照4倍梯度稀释,每个孔加50 μl,于37℃孵育1h后,用0.05% PBST冲洗3次;
4)加入HRP标记的SA(在0.05% PBST中0.1 μg/ml,100 ul/well),于37℃反应30min后,使用0.05% PBST冲洗6次;
5)使用3,3′,5,5′-四甲基联苯胺(TMB)作为快速显色指示剂,反应10 min;
6)将所述步骤5)的显色反应经终止液终止,用酶标仪在波长为OD 450-630 nm下测量,得到吸光度OD值,并绘制拟合曲线,根据该结果进行血液安全性评估。
具体地,所述待测抗体为TfR1抗体,可以是单克隆抗体、双特异性抗体、多特异性抗体、或抗体片段,可以是具有特定组织靶向性的长度不少于10个氨基酸的蛋白质,可以是环状或线性的多肽。
具体地,所述试剂X为试剂R2或试剂R3。其中,当所述试剂X为R2时,若检测结果仅得到吸光度OD值表,却无法得到IC50曲线,则说明待测样品不和转铁蛋白(Transferrin,Tf)识别相同抗原结合位点,因此该样品具备血液安全性;若检测结果能得到IC50曲线,则说明待测样品和转铁蛋白(Tf)识别相同抗原结合位点,表明该样品不具备血液安全性。其中,当所述试剂X为R3(和Human Transferrin R/CD71抗原结合,但不和Transferrin(Tf)识别相同抗原结合位点)时,若检测结果能得到IC50曲线,则说明待测样品不和Transferrin(Tf)识别相同抗原结合位点,且与TfR1的结合力比R3更强,为安全的优选抗体,反之,如果未检测到IC50曲线,则说明待测样品与TfR1的结合力比R3弱,该样品不是优选抗体。
以下通过具体实施例对本申请进行进一步描述。
实验所用材料:
试剂R1(Human Transferrin R/CD71抗原),购自Bio-Techne China Co. Ltd.,货号2474-TR;
试剂R2(AF2474),购自Bio-Techne China Co. Ltd.,货号DB0105615;
试剂R3,委托南京金斯瑞蓬勃生物有限公司生产;
包被缓冲液CBS(0.1mol/L碳酸盐缓冲液,pH9 .6),取3.34g无水碳酸氢钠和1g无水碳酸钠,加蒸馏水充分搅拌,调节pH值至9.6,定容至500mL备用。
稀释液(0 .01mol/L的磷酸盐缓冲液,pH 7 .4),称取7.9g NaCl、0.2g KCl、0.24gKH2PO4和1.8g K2HPO4,溶于800ml蒸馏水中,用HCl调节溶液的pH至7.4,最后加蒸馏水定容至1L,保存于4℃冰箱中备用;
底物显色液(双组分TMB显色液),购自北京索莱宝科技有限公司,货号PR1210;
终止液为1 M 盐酸溶液;
洗涤液(0.05% PBST),含0.05%Tween20(v/v)的0.01M pH 7.4 PBS缓冲液。
若未特别说明,以下实施例中所使用的实验试剂均为本领域常规试剂,可按照本领域常规方法配制而得或商购获得。若未特别说明,以下实施例中所使用的实验方法,均为本领域常规方法,可参考相关实验手册,例如分子克隆实验手册(Sambrook J&Russell DW,Molecular cloning: a laboratory manual, 2001),或制造厂商说明书。除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域普通技术人员通常理解的含义相同的含义。
实施例1 用于评价TfR1抗体血液安全性的试剂盒验证1:
1)将1 μg/ml的未经Biotin标记的试剂R1(Human Transferrin R/CD71抗原,CBS,100 μL/well)于4℃包被于酶标板中过夜孵育,经0.05% PBST缓冲液洗涤后,经3% MPBS缓冲液于37℃封闭1h,再经0.05% PBST冲洗;
2)将30 μg/mL的试剂R2(AF2474),按照4倍梯度稀释,每个孔加50 μL,加入封闭后的酶标板;
3)再加入Biotin标记的转铁蛋白(Tf)0.032 μg/ml(50 μL/well),于37℃孵育1h后,用0.05% PBST冲洗3次;
4)使用HRP标记的SA(在0.05% PBST中0.1 μg/ml,100 ul/well),于37℃反应30min后,使用0.05% PBST冲洗6次;
5)使用3,3’,5,5’-四甲基联苯胺(TMB)作为快速显色指示剂,1 μg/mL的转铁蛋反应10 min;
6)将所述步骤5)的显色反应经终止液1M 盐酸终止,用酶标仪在波长为OD 450-630 nm下测量,得到吸光度OD值(表1),并绘制拟合曲线(结果见图1)。
表1:
。
根据吸光度值(表1中的A对应的值)制作的拟合曲线,可得到IC50值为2.845,对应的结果表明:R2在>1 μg/mL(~10nM)条件下能够阻断Tf和TfR1之间的结合。
实施例2 用于评价TfR1抗体血液安全性的试剂盒验证2:
1)将1 μg/mL的转铁蛋白(Tf)的酶标板,于4℃包被于酶标板中过夜孵育,经0.05%PBST缓冲液洗涤后,经3% MPBS缓冲液于37℃封闭1h,再经0.05% PBST冲洗;
2)将30 μg/mL的试剂R2(AF2474),按照4倍梯度稀释,每个孔加50 μL,加入封闭后的酶标板;
3)再加入Biotin标记的试剂R1(Human Transferrin R/CD71抗原)0.06 μg/ml(50uL/well),于37℃孵育1h后,用0.05% PBST冲洗3次;
4)使用HRP标记的SA(在0.05% PBST中0.1 μg/ml,100 ul/well),于37℃反应30min后,使用0.05% PBST冲洗6次;
5)使用3,3’,5,5’-四甲基联苯胺(TMB)作为快速显色指示剂,反应10 min;
6)将所述步骤5)的显色反应经终止液终止,在波长为OD 450-630 nm下测量,得到吸光度OD值(表2),并绘制拟合曲线(结果请见图2)。
表2:
。
根据吸光度值制作的拟合曲线,可得到IC50值为2.862,即AF2474在>1ug/mL(~10nM)下能够阻断Tf和TfR1之间的结合。
实施例1和实施例2结果类似,证明了先包被R1和先包被Tf检测结果相同。
实施例3 用于评价TfR1抗体血液安全性的试剂盒(二)验证1:
1)将1 μg/mL的Transferrin(Tf)的酶标板,于4℃包被于酶标板中过夜孵育,经0.05% PBST缓冲液洗涤后,经3% MPBS缓冲液于37℃封闭1h,再经0.05% PBST冲洗;
2)将120 μg/ml的试剂R3(在PBST中),按照2倍梯度稀释,每个孔加50 μL,加入封闭后的酶标板;
3)再加入Biotin标记的R1(Human Transferrin R/CD71抗原)0.06 μg/ml(50 uL/well),于37℃孵育1h后,用0.05% PBST冲洗3次;
4)使用HRP标记的SA(0.1μg/ml in 0.05% PBST,100 ul/well),于37℃反应30min后,使用0.05% PBST冲洗6次;
5)使用3,3’,5,5’-四甲基联苯胺(TMB)作为快速显色指示剂,反应10 min;
6)将所述步骤5)的显色反应经终止液终止,在波长为OD 450-630 nm下测量,得到吸光度OD值(表3)。
表3:
。
根据得到的吸光度OD值表,无法得到IC50曲线。说明虽然R3是和HumanTransferrin R/CD71抗原结合的抗体,但其无法将Tf从R1上竞争下来,因此其不和Transferrin(Tf)识别相同抗原结合位点。
实施例4 用本申请的试剂盒对待测抗体安全性进行评估:
1)将1 μg/ml的试剂R2(AF2474,100 ul/well)于4℃包被于酶标板中过夜孵育,经0.05% PBST缓冲液洗涤后,经3% MPBS缓冲液于37℃封闭1h,再经0.05% PBST冲洗;
2)将30 μg/ml的试剂R1(Biotin标记的Human Transferrin R/CD71抗原, 在CBS中, 50 μl/well)加入封闭后的酶标板;
3)加入15 μg/ml的待测抗体Y,按照4倍梯度稀释,每个孔加50 μl,于37℃孵育1h后,用0.05% PBST冲洗3次;
4)加入HRP标记的SA(在0.05% PBST中0.1 μg/ml,100 ul/well),于37℃反应30min后,使用0.05% PBST冲洗6次;
5)使用3,3′,5,5′-四甲基联苯胺(TMB)作为快速显色指示剂,反应10 min;
6)将所述步骤5)的显色反应经终止液终止,用酶标仪在波长为OD 450-630 nm下测量,得到吸光度OD值(表4)。根据得到的吸光度OD值表,无法得到IC50曲线。
将试剂R2替换为试剂R3,重复上述步骤,获得吸光度OD值(表5),绘制拟合曲线,如图3中所示,根据该结果进行血液安全性评估。
表4:
。
表5:
。
从实验结果可以看出,当使用试剂R1和试剂R2进行评估时,检测结果仅得到吸光度OD值表,无法得到IC50曲线;当使用试剂R1和试剂R3进行评估时,检测结果能得到IC50曲线。因此,待测抗体Y的血液安全性良好,不会影响转铁蛋白Tf和TfR1的结合,且与TfR1的结合力比R3更强,为安全的优选抗体。
以上3个实施例利用转铁蛋白(Tf)溶液对试剂盒进行性能验证,1个实施例检测了待测抗体Y的血液安全性,结果表明,试剂盒能明显的检测出待测抗体是否影响TfR1和Tf结合进一步影响Fe的转运,从而得出TfR1抗体血液安全性评价的结果。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种用于进行TfR1抗体血液安全性评估的试剂盒,所述试剂盒包括试剂R1和试剂X,其中,所述试剂R1为标记的Human Transferrin R/CD71抗原;所述试剂X为R2或R3,其中,所述试剂R2为AF2474,其能够与Human Transferrin R/CD71抗原结合,并与Tf识别相同的抗原结合位点;所述试剂R3为能够与Human Transferrin R/CD71抗原结合、但不与Tf识别相同抗原结合位点的抗体。
2.根据权利要求1所述的试剂盒,其特征在于,所述试剂盒还包含样品稀释液、底物显色液、终止液和/或浓缩洗涤液。
3.根据权利要求2所述的试剂盒,其特征在于,所述底物显色液为双组分TMB显色液。
4.根据权利要求2所述的试剂盒,其特征在于,所述样品稀释液为pH 9.6的碳酸盐缓冲液,所述浓缩洗涤液为含0.05%吐温20的pH 7.4的磷酸盐缓冲液。
5.一种使用权利要求1-4中任一项所述的试剂盒进行TfR1抗体血液安全性评估的方法,所述方法包括:
1)将试剂X包被于酶标板中;
2)将试剂R1加入酶标板;
3)按照梯度稀释依次加入待测抗体;
4)加入HRP标记的SA;
5)加入快速显色指示剂;
6)测量吸光度OD值,绘制曲线,进行评估。
6.根据权利要求5所述的方法,其特征在于,所述待测抗体为TfR1的单克隆抗体、多特异性抗体、或抗体片段。
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