CN113009135B - 一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用 - Google Patents
一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种检测CD47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用。所述试剂盒包括磁微粒‑免疫球蛋白结合分子‑CD47抗体复合物、CD47抗体‑中性亲和素‑生物素‑辣根过氧化物酶复合物、样品稀释液、发光液A、发光液B、洗涤剂、CD47标准品梯度溶液、反应管及使用说明书。本发明还提供的管式磁微粒化学发光免疫定量试剂盒的制备方法与应用。本发明将磁性分离技术、化学发光检测技术与免疫学方法三者相结合,得到的式磁微粒化学发光免疫定量试剂盒检测的精密性和稳定性得到极大的提升。并且本发明试剂盒稳定性好,在2~8℃至少可以稳定存放一年。
Description
技术领域
本发明涉及免疫分析医学技术领域,具体的,本发明提供了一种检测CD47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用。
背景技术
CD47属于免疫球蛋白超家族成员,由于最初与整合素ανβ3蛋白结合和共纯化而被发现,因此又名整合素相关蛋白(integrin associated protein,IAP),相对分子质量为50kD,CD47的结构包括:一个单独的Ig V样的N末端,五个高度疏水延伸的跨膜片段和一个短的选择性拼接的羧基段胞质C末端,其广泛表达于不同组织细胞表面,如造血细胞(红细胞、淋巴细胞、血小板等)、非造血细胞(胎盘、肝和脑细胞等)及肿瘤细胞等。CD47可与信号调节蛋白α(Signal regulatory proteinα,SIRPα)、血小板反应蛋白(thrombospondin-1,TSP1)以及整合素(integrins)相互作用,介导凋亡、增殖、免疫等一系列的反应,例如在年轻的红细胞上CD47表达相对较高,而在衰老的红细胞上CD47表达下调,使得衰老的红细胞被识别和清除;当CD47与SIRPα结合时,可帮助巨噬细胞识别“非我”和“本我”细胞,肿瘤细胞通过高表达CD47分子传导抑制性信号,即“别吃我”信号,阻止T淋巴细胞和巨噬细胞激活,帮助肿瘤细胞逃逸。
正常生理情况下,原癌细胞同时表达促进吞噬细胞吞噬的钙网蛋白和抑制吞噬的CD47,两者处于动态平衡;病理状态下,CD47表达增加,抑制钙网蛋白介导的吞噬作用,使肿瘤细胞逃脱免疫监视。研究显示,CD47在多种人类肿瘤细胞中表达上调,即无论是血液系统肿瘤还是实体瘤,如肺癌、乳腺癌、胃癌、膀胱癌、急性淋巴细胞白血病等,肿瘤细胞膜上的CD47都呈现出过表达状态,故CD47可作为诊断肿瘤及判定预后的较可靠指标。
研究发现,非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中也发现CD47表达显著上调,且越高表达的患者预后不良。研究表明CD47高表达的肿瘤患者生存期较短。另外,肿瘤组织中CD47表达与肺癌患者TNM高分期及术后化疗抵抗均显著相关,且研究结果显示,血清CD47高表达的患者较低表达者具有更高的TNM分期及更低的细胞分化程度,且血清CD47表达水平与患者TNM分期正相关,与患者细胞分化程度呈负相关,提示CD47参与NSCLC患者病情的进展,并与细胞恶性转化密切相关。NSCLC患者血清CD47表达水平与肿瘤进展、细胞恶性程度及放化疗抵抗密切相关,是较可靠的NSCLC临床标志物。
目前,对CD47蛋白的体外检测方法研究较少,或是仅处于科学研究,因此急需开发一种能够快速、灵敏、准确地对CD47蛋白表达水平进行检测的试剂盒。
发明内容
针对现有技术的不足,本发明提供一种检测CD47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用,为CD47在临床诊断中寻求较快速、灵敏的诊断方法。
本发明的技术方案如下:
一种检测CD47的管式磁微粒化学发光免疫定量试剂盒,包括磁微粒复合物、样品稀释液和酶结合物;
所述的磁微粒复合物为磁微粒-免疫球蛋白结合分子-CD47抗体复合物;所述的酶结合物为CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物;所述的CD47抗体为CD47单克隆抗体;
所述样品稀释液的配方是:除菌的1L纯化水中,含NaCl 7~8g,NaH2PO4·2H2O0.15~0.25g,NaH2PO4·2H2O 2.5~3.0g,酪蛋白钠盐3.5~4.0g,Proclin300 1.0~2.0mL。
根据本发明优选的,所述检测CD47的管式磁微粒化学发光免疫定量试剂盒还包括:发光液A、发光液B、20×浓缩洗涤剂和CD47标准品梯度溶液。
根据本发明优选的,所述的磁微粒的结构为:内层为F3O4磁性材料,外层为覆盖F3O4磁性材料的聚合物,微粒总粒径为0.8μm~1.5μm;
根据本发明优选的,所述的磁微粒按照如下方法制备:在68~75℃,N2保护下将无水乙醇(EtOH)、H2O、聚乙二醇-6000(PEG)、F3O4加入到反应器中,快速搅拌0.5~1h;然后将过氧化苯甲酰(BPO)溶解在苯乙烯(St)中缓慢滴加进反应器中,再依次加丙烯酸(AA),二乙烯基苯(DVB),于快速搅拌下恒温反应10~15h后,采用H2O、无水乙醇(EtOH)和稀HCl反复洗涤3~5次,经分离后得到羧基磁微粒;
所述无水乙醇、H2O、聚乙二醇-6000、F3O4、过氧化苯甲酰、苯乙烯、丙烯酸和二乙烯基苯的质量比为(35~40):(30~34):(5~6):(1~2):(5~6):(12~15):(1~2):(1~2)。
根据本发明优选的,所述免疫球蛋白结合分子为葡萄球菌蛋白A、链球菌蛋白G或大消化链球菌蛋白L中的一种或两种以上的混合物。
根据本发明优选的,所述发光液A中含有鲁米诺3.5~4.5mmol/L,对碘酚0.3~0.8mmol/L,FeCl3 9~30μmol/L,二氧六烷1~5mL/L,Tris-HCl缓冲液5mmol/L,pH 8.6,避光保存。
根据本发明优选的,所述发光液B为浓度为1~10mmol/L的过氧化脲溶液。
根据本发明优选的,所述洗涤剂中含有0.3~0.5mol/L磷酸氢二钠,0.04~0.06mol/L磷酸二氢钠,2.5~3.5mol/L氯化钠,8~12mL/L Tween-20,0.1~0.3mL/LProclin300。
根据本发明优选的,所述CD47标准品梯度溶液有10个浓度的溶液,以如下方法制得:按照3~5wt%牛血清白蛋白、0.85wt%氯化钠、3~5wt%海藻糖、pH7.3~7.5的1.0mmol/L Na2HPO4-NaH2PO4的缓冲液、0.1~0.3mL/L Proclin300配制标准品稀释液,再用标准品稀释液制备400、200、50、25、5、1、0.5、0.1、0.05、0ng/mL 10个浓度梯度的CD47标准品溶液,其中0ng/mL为标准品稀释液。
本发明所述试剂盒还包括反应管,反应管的材料为透明的聚苯乙烯、聚乙烯、聚丙烯、聚氯乙烯或玻璃。
本发明所述试剂盒还包括使用说明书。
一种上述检测CD47的管式磁微粒化学发光免疫定量试剂盒的制备方法,包括步骤如下:
S1:磁微粒-免疫球蛋白结合分子-CD47抗体复合物的制备
将400~500μg羧基磁微粒置于在MES(50mM,pH 5)缓冲液中,然后添加100~120μg的EDC和100~120μg Sulfo-NHS后,室温震动孵育40~45min,离心洗涤后,加80~120μg免疫球蛋白结合分子孵育3~3.5h,再离心洗涤,加入80~120μg CD47单克隆抗体与磁微粒-免疫球蛋白结合分子孵育3~3.5h,得到磁微粒-免疫球蛋白结合分子-CD47抗体复合物;
S2:CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的制备
1)CD47抗体-中性亲和素的制备
将15~60mg中性亲和素溶解在100mL 0.1mol/L磷酸盐缓冲液中,加入0.5~0.8mLCD47抗体溶液(0.2mg/mL),再逐滴加50mL的0.1~0.3%戊二醛溶液(0.1mol/L磷酸钠缓冲液,pH 6.8)中,避光搅拌16~20h,然后在0.1mol/L磷酸钠缓冲液(pH 7.4)中透析除去戊二醛,透析完成后的溶液中加入等体积的丙三醇,得到CD47抗体-中性亲和素;
2)生物素标记的辣根过氧化物酶结合物的制备
取4~6mg辣根过氧化物酶溶于1mL蒸馏水中,加入过碘酸钠溶液0.2mL(4.3mg),室温避光搅拌20min,用醋酸缓冲液(0.01mol/L pH4.4)进行过夜透析提纯后,加入0.1~0.5mg生物素,用碳酸盐缓冲液(0.2mol/L pH9.5)调pH到8.0~9.0左右,室温避光振摇2~2.5h,得到生物素标记的辣根过氧化物酶结合物;
3)CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的结合
将CD47抗体-中性亲和素用PBS洗涤,加入用PBS稀释10~50倍体积生物素标记的辣根过氧化物酶结合物,在4℃下孵育1~2h,得到CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物;
S3:组装
将磁微粒-免疫球蛋白结合分子-CD47抗体复合物、CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物、样品稀释液、发光液A、发光液B、洗涤剂、CD47标准品梯度溶液和反应管及使用说明书组装成盒,即得到检测CD47的管式磁微粒化学发光免疫定量试剂盒,储存于2~8℃。
上述管式磁微粒化学发光免疫定量试剂盒在检测CD47中的应用。
本发明还提供上述管式磁微粒化学发光免疫定量试剂盒检测CD47的方法,包括步骤如下:
(1)将待测样品及试剂盒内的试剂,于20~25℃平衡25±5min,将洗涤液用蒸馏水稀释20倍后备用;
(2)设置标准品孔和待测样品孔,分别向待测样品孔和标准品孔中加入50μL磁微粒复合物悬浮液,接着分别加入50μL待测样品和50μL CD47标准品梯度溶液后,再加入50μL样品稀释液,然后在37℃下温浴,震荡10min;
(3)将反应管放置在磁分离器上静置10~15s后,弃去液体,将反应管从磁分离器上取下,用洗涤剂冲洗磁微粒复合物,洗涤完成后进行磁分离,弃上清,按此方式重复洗涤5~8次,每次洗涤时浸泡15~20s,每孔加洗涤液量不少于600μL;
(4)继续加入100μL CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物,在37℃下温浴,震荡5min;
(5)重复步骤(3)1~2次;
(6)继续加入150μL发光液A,再加入150μL发光液B,室温避光4min后测定发光强度;
(7)以标准品的各浓度为横坐标,RLU值为纵坐标,用标准品的浓度与RLU值绘制出标准曲线,根据待测样品的RLU值,计算出样品的浓度,如进行了稀释,则根据稀释倍数,计算出样品的实际浓度。
本发明的原理:采用双抗体夹心法测定样本中的CD47,在免疫球蛋白结合分子-磁微粒悬浮液中,加入CD47-抗体,通过免疫球蛋白结合分子可定向结合CD47抗体的Fc端,形成磁微粒-免疫球蛋白结合分子-CD47-抗体-CD47抗原-CD47抗体-中性亲和素-生物素-HRP复合物,用外加磁场将复合物吸附在反应管底部,清洗掉游离的成分,加入底物工作液,在氧化剂和增强剂作用下,HRP催化鲁米诺生成处于激发态的氨基邻笨二甲酸离子,其恢复待基态时,释放出425nm的光子,于第4分钟测定各加样孔的发光值RLU。待测样品的RLU与样本CD47浓度呈正相关。待测样品中的CD47浓度依据由标准品CD47浓度和对应的RLU绘制标准曲线(标准品浓度为横坐标,对应的发光值为纵坐标)进行定量,从而得出待测样品中的CD47含量。
有益效果
1、本发明提供的管式磁微粒化学发光免疫定量试剂盒可快速有效地进行样本中CD47含量的检测,采用的免疫球蛋白结合分子可定向结合CD47抗体的Fc端,不影响抗体与抗原的特异性结合,中性亲和素-生物素使得酶标记的信号放大更充分。使用过程稳定、无放射性污染,在没有外加磁场存在的情况下,磁性微粒悬浮在液体中,使得抗原抗体反应类似于均相反应,当磁性微粒在外加磁场作用时,可快速地分离,方便洗涤;使得包被固相接近于液相状态,检测的精密性和稳定性得到极大的提升。并且本发明试剂盒稳定性好,在2~8℃至少可以稳定存放一年。
2、本发明将磁性分离技术、化学发光检测技术与免疫学方法三者相结合,综合了化学发光灵敏度高、线性范围广、测定速度快;免疫法的特异性高、准确性好;磁微粒可扩大传统板式的固相载体面积,且在磁场可控运动的特点情况下,可实现待测物的快速富集及分离,具有更快的检测速度,使用本发明提供的检测方法约30min即可得出结果。
附图说明
图1为实施例4检测CD47的管式磁微粒化学发光免疫定量试剂盒线性分析曲线图。
图2为实施例4检测CD47的管式磁微粒化学发光免疫定量试剂盒干扰因素分析曲线图;
图中:(a)为血红蛋白曲线图,(b)为甘油三酯曲线图,(c)为胆固醇曲线图。
图3为对比例1检测CD47的管式磁微粒化学发光免疫定量试剂盒线性分析曲线图。
图4为对比例2检测CD47的管式磁微粒化学发光免疫定量试剂盒线性分析曲线图。
具体实施方式
下述实施例中,本发明中未详细说明的方法均为本领域技术人员所熟知的。本发明中涉及的原料或试剂均可以在市场上购买或是根据现有技术制备得到。
实施例1
一种检测CD47的管式磁微粒化学发光免疫定量试剂盒,包括磁微粒复合物、样品稀释液、酶结合物、发光液A、发光液B、20×浓缩洗涤剂、CD47标准品梯度溶液、反应管及使用说明书;
其中,所述的磁微粒复合物为磁微粒-免疫球蛋白结合分子-CD47抗体复合物;所述的酶结合物为CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物;所述的CD47抗体为生物素标记的CD47单克隆抗体;所述免疫球蛋白结合分子为葡萄球菌蛋白A。
所述样品稀释液的配方是:除菌的1L纯化水中,含NaCl 8g,NaH2PO4·2H2O 0.2g,NaH2PO4·2H2O 2.5g,酪蛋白钠盐3.5g,Proclin300 1.0mL。
所述发光液A中含有鲁米诺4mmol/L,对碘酚0.5mmol/L,FeCl3 15μmol/L,二氧六烷3mL/L,Tris-HCl缓冲液5mmol/L,pH 8.6,避光保存。
所述发光液B为浓度为5mmol/L的过氧化脲溶液。
所述洗涤剂中含有0.4mol/L磷酸氢二钠,0.05mol/L磷酸二氢钠,2.5mol/L氯化钠,8mL/L Tween-20,0.2mL/L Proclin300。
所述CD47标准品梯度溶液有10个浓度的溶液,以如下方法制得:按照4wt%牛血清白蛋白、0.85wt%氯化钠、4wt%海藻糖、pH7.4的1.0mmol/L Na2HPO4-NaH2PO4的缓冲液、0.2mL/L Proclin300配制标准品稀释液,再用标准品稀释液制备400、200、50、25、5、1、0.5、0.1、0.05、0ng/mL 10个浓度梯度的CD47标准品溶液,其中0ng/mL为标准品稀释液。
所述的磁微粒的结构为:内层为F3O4磁性材料,外层为覆盖F3O4磁性材料的聚合物,微粒总粒径为1μm;
具体制备方法如下:
在72℃,N2保护下将无水乙醇(EtOH)、H2O、聚乙二醇-6000(PEG)、F3O4加入到反应器中,快速搅拌0.6h;然后将过氧化苯甲酰(BPO)溶解在苯乙烯(St)中缓慢滴加进反应器中,再依次加丙烯酸(AA),二乙烯基苯(DVB),于快速搅拌下恒温反应11h后,采用H2O、无水乙醇(EtOH)和稀HCl反复洗涤6次,经分离后得到磁微粒;
所述无水乙醇、H2O、聚乙二醇-6000、F3O4、过氧化苯甲酰、苯乙烯、丙烯酸和二乙烯基苯的质量比为38:33:5.5:1.7:5.8:13:1.3:1.7。
实施例2
一种检测CD47的管式磁微粒化学发光免疫定量试剂盒的制备方法,包括步骤如下:
S1:磁微粒-免疫球蛋白结合分子-CD47抗体复合物的制备
将500μg羧基磁微粒置于在MES(50mM,pH5)缓冲液中,然后添加120μg的EDC和120μg Sulfo-NHS后,室温震动孵育45min,离心洗涤后,加120μg葡萄球菌蛋白A孵育3.5h,再离心洗涤,加120μgCD47单克隆抗体与磁微粒-免疫球蛋白结合分子孵育3.5h,得到磁微粒-免疫球蛋白结合分子-CD47抗体复合物;
S2:CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的制备
1)CD47抗体-中性亲和素的制备
将40mg中性亲和素溶解在100mL 0.1mol/L磷酸盐缓冲液中,加入0.6mL CD47抗体溶液(0.2mg/mL),再逐滴加50mL的0.2%戊二醛溶液(0.1mol/L磷酸钠缓冲液,pH 6.8)中,避光搅拌18h,然后在0.1mol/L磷酸钠缓冲液(pH 7.4)中透析,4℃下透析提纯8h(换液5次),除去戊二醛,透析完成后的溶液中加入等体积的丙三醇,得到CD47抗体-中性亲和素;-20℃保存;
2)生物素标记的辣根过氧化物酶结合物的制备
取5mg辣根过氧化物酶溶于1mL蒸馏水中,加过碘酸钠溶液0.2mL(4.3mg),室温避光搅拌20min,由暗红色变成绿色,说明氧化完成;用醋酸缓冲(0.01mol/L pH4.4)液透析过夜后,加入0.3mg生物素,用碳酸盐缓冲液(0.2mol/L pH9.5)调pH到8.5,室温避光振摇2小时,得到生物素标记的辣根过氧化物酶结合物;
3)CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的结合
将CD47抗体-中性亲和素用PBS洗涤,加入用PBS稀释35倍体积的生物素标记的辣根过氧化物酶结合物,在4℃下孵育1.5h,制成CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物,4℃无菌保存;
S3:组装
将磁微粒-免疫球蛋白结合分子-CD47抗体复合物、CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物、样品稀释液、发光液A、发光液B、洗涤剂、CD47标准品梯度溶液、反应管及使用说明书组装成盒,即得到检测CD47的管式磁微粒化学发光免疫定量试剂盒,储存于2~8℃。
实施例3
实施例所述的管式磁微粒化学发光免疫定量试剂盒在检测CD47中的应用,检测方法如下:
(1)将待测样品及试剂盒内的试剂,于20~25℃平衡25±5min,将洗涤液用蒸馏水稀释20倍后备用;
(2)设置标准品孔和待测样品孔,分别向待测样品孔和标准品孔中加入50μL磁微粒复合物悬浮液,接着分别加入50μL待测样品和50μL CD47标准品梯度溶液后,再加入50μL样品稀释液,然后在37℃下温浴,震荡10min;
(3)将反应管放置在磁分离器上静置10s后,弃去液体,将反应管从磁分离器上取下,用洗涤剂冲洗磁微粒复合物,洗涤完成后进行磁分离,弃上清,按此种方式洗涤6次,每次洗涤时浸泡20s,每孔加洗涤液量为650μL;
(4)继续加入100μL CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物,在37℃下温浴,震荡5min;
(5)重复步骤(3)1次;
(6)继续加入150μL发光液A,再加入150μL发光液B,室温避光4min后测定发光强度;
(7)以标准品的各浓度为横坐标,RLU值为纵坐标,用标准品的浓度与RLU值绘制出标准曲线,根据待测样品的RLU值,计算出样品的浓度,如进行了稀释,则根据稀释倍数,计算出样品的实际浓度。
实施例4试剂盒性质分析
1)物理检查:
结果:液体组分澄清,无沉淀或絮状物;其他组分无包装破损。
2)标准曲线的线性:
采用CD47标准品的10个浓度梯度,梯度浓度为400、200、50、25、5、1、0.5、0.1、0.05、0ng/mL的CD47标准品溶液,用实施例3的检测方法进行标准品溶液的RLU值读数,线性分析读数为表1所示。绘制标准曲线见图1。
表1试剂盒线性分析读数
由图1可知,曲线方程为:Y=1462X-1123.8,R2=0.9997,可见在0~400ng/mL范围内,本试剂盒线性良好。
3)分析灵敏度:
20个零标准品浓度RLU的平均值加上两个标准差,计算相应的可检测浓度,结果统计见表2。
表2分析灵敏度试验结果
由表2可知,带入图1中的曲线方程,即可计算出CD47浓度,即可检测人CD47最低检测线为0.81ng/mL。
4)准确性:
以样本回收率进行验证,根据《体外诊断试剂分析性能评估(准确度-回收实验)技术审查指导原则》进行实验操作。结果统计见表3。
表3回收率实验结果
如表3所述,添加回收率的范围应在90.0%~110.0%,本发明检测的添加回收率在98.0%~107.0%,平均添加回收率为102.5%,符合要求。
5)精密度:
用本发明对CD47低、中、高浓度的三个样本分别进行检测,在同一次实验中每个样本平行做10次。计算批内精密度(CV);将3个样本每天检测1次,连续测定10d,计算批间精密度(CV)。结果统计见表4。
表4精密性试验结果
如表5所述,低、中、高浓度样本检测结果的变异系数均<10%,说明该方法具有良好的重复性。
6)特异性:血清癌胚抗原(CEA)、血清甲胎蛋白(AFP)、前列腺特异抗原(PSA)、绒毛膜促性腺激素(HCG)是目前比较公认的常见肿瘤标志物,因此需要对CD47检测方法受血清中CEA、AFP、PSA、HCG的影响进行评价。
表5与其他血清标志物的交叉反应率
样品 | 实际浓度 | 测定值 | 交叉反应率(%) |
CEA | 25ng/mL | 0.0375ng/mL | 0.15 |
AFP | 1200ng/mL | 2.88ng/mL | 0.24 |
PSA | 23ng/mL | 0.0391ng/mL | 0.17 |
HCG | 45ng/mL | 0.072ng/mL | 0.16 |
如表5所示,用本发明检测方法分别检测浓度为25ng/mL、的CEA、AFP、PSA、HCG血清,交叉反应率分别只有0.15%、0.24%、0.17%、0.16%,表明本检测方法特异性很高。
7)其他干扰因素的影响
如图2所示,用本方法检测不同浓度的干扰因素,结果显示,血红蛋白浓度不大于3000mg/L、胆固醇浓度不大于10g/L、甘油三脂浓度不大于35mmol/L时,对CD47测定结果也没有影响(P>0.5)。
8)稳定性:
结果:将试剂盒中各试剂组分于37℃下放置7d无性状变化,稳定性良好。
对比例1
与本发明所述的检测CD47的管式磁微粒化学发光免疫定量试剂盒的制备方法不同处为,磁微粒复合物为磁微粒-CD47抗体复合物,其余相同。
磁微粒-CD47抗体复合物制备方式如下:
将500μg羧基磁微粒置于在MES(50mM,pH5)缓冲液中,然后添加120μg的EDC和120μg Sulfo-NHS后,室温震动孵育45min,离心洗涤后,加120μg CD47单克隆抗体与磁微粒-免疫球蛋白结合分子孵育3.5h。
对比例1标准曲线的线性:
采用CD47标准品的10个浓度梯度,梯度浓度为400、200、50、25、5、1、0.5、0.1、0.05、0ng/mL的CD47标准品溶液,用实施例3的检测方法进行标准品溶液的RLU值读数,线性分析读数为表6所示。绘制标准曲线见图3。
表6试剂盒线性分析读数
由图3可知,曲线方程为:Y=1312.6X+15338,R2=0.9368,可见在0~400ng/mL范围内,试剂盒线性较差。
对比例2
与本发明所述的检测CD47的管式磁微粒化学发光免疫定量试剂盒的制备方法不同处为,酶结合物为CD47抗体-辣根过氧化物酶结合物,其余相同。
CD47抗体-辣根过氧化物酶结合物制备方式如下:
取5mg辣根过氧化物酶溶于1mL蒸馏水中,加过碘酸钠溶液0.2ml(4.3mg),室温避光搅拌20min,由暗红色变成绿色,说明氧化完成;用醋酸缓冲(0.01mol/L pH4.4)液透析过夜后,加入1.4mg CD47抗体,用碳酸盐缓冲液(0.2mol/L pH9.5)调pH到8.5,于4℃作用2h。
对比例2标准曲线的线性:
采用CD47标准品的10个浓度梯度,梯度浓度为400、200、50、25、5、1、0.5、0.1、0.05、0的CD47标准品溶液,用实施例3的检测方法进行标准品溶液的RLU值读数,线性分析读数为表7所示。绘制标准曲线见图4。
表7试剂盒线性分析读数
由图4可知,曲线方程为:Y=1324.1X+11222,R2=0.958,可见在0~400ng/mL范围内,试剂盒线性较差。
试验例
研究对象及方法:106例同步放化疗的非小细胞肺癌(NSCLC)患者,所有患者均经纤维支气管镜或CT引导下穿刺活检确诊为NSCLC,且均为初发。收集患者临床资料,包括性别、年龄,其中男86例,女20例;年龄36~75岁,平均(56.2±12.0)岁。所有患者均行三维适形放疗或调强放疗,采用胸部CT定位扫描仪对患者放疗区域进行扫描,将扫描好的图像进行勾画,并制订放疗。根据放化疗疗效将患者分为敏感组和抵抗组,比较两组患者各时期血清CD47表达水平的差异。
检测方法:别于治疗前、治疗4周及治疗结束后,采取所有患者空腹状态肘静脉血3ml并置于采血试管中,凝集30分钟后置于超低温离心器,3000r/min离心10分钟,取上清液,-80℃冰箱保存。取得的样本经本发明的检测试剂盒进行测定。结果统计见表8。
表8不同时间点不同疗效的患者血清CD47表达水平比较(ng/mL)
组别 | 治疗前 | 治疗4周 | 治疗结束时 |
敏感组(n=35) | 47.09±2.87 | 38.59±2.58 | 17.81±4.20 |
抵抗组(n=71) | 73.63±3.77 | 54.99±2.73 | 33.32±3.18 |
注:与同一时间点敏感组相比,*P<0.05;与本组治疗前相比,#P<0.05。
如表8所示,治疗结束时,敏感组和抵抗组患者血清CD47表达水平均显著低于治疗前(P<0.05);敏感组患者在治疗前、治疗4周、治疗结束时血清CD47表达水平均显著低于抵抗组(P<0.05),以上说明,本发明通过血清CD47的检测可对NSCLC患者病情进展进行评估,并对后期诊疗方案提供指导。
Claims (1)
1.一种检测CD47的管式磁微粒化学发光免疫定量试剂盒,其特征在于,包括磁微粒复合物、样品稀释液和酶结合物;
所述的磁微粒复合物为磁微粒-免疫球蛋白结合分子-CD47抗体复合物;所述的酶结合物为CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物;所述的CD47抗体为CD47单克隆抗体;
所述样品稀释液的配方是:除菌的1L纯化水中,含NaCl 7~8g,NaH2PO4·2H2O 0.15~0.25g,NaH2PO4·2H2O 2.5~3.0g,酪蛋白钠盐3.5~4.0g,Proclin300 1.0~2.0mL;
所述检测CD47的管式磁微粒化学发光免疫定量试剂盒还包括:发光液A、发光液B、20×浓缩洗涤剂、CD47标准品梯度溶液、反应管和使用说明书;
所述的磁微粒的结构为:内层为F3O4磁性材料,外层为覆盖F3O4磁性材料的聚合物,微粒总粒径为0.8μm~1.5μm;
所述的磁微粒按照如下方法制备:在68~75℃,N2保护下将无水乙醇、H2O、聚乙二醇-6000、F3O4加入到反应器中,快速搅拌0.5~1h;然后将过氧化苯甲酰溶解在苯乙烯中缓慢滴加进反应器中,再依次加丙烯酸,二乙烯基苯,于快速搅拌下恒温反应10~15h后,采用H2O、无水乙醇和稀HCl反复洗涤3~5次,经分离后得到磁微粒;
所述的无水乙醇、H2O、聚乙二醇-6000、F3O4、过氧化苯甲酰、苯乙烯、丙烯酸和二乙烯基苯的质量比为(35~40):(30~34):(5~6):(1~2):(5~6):(12~15):(1~2):(1~2);
所述免疫球蛋白结合分子为葡萄球菌蛋白A;
所述发光液A中含有鲁米诺3.5~4.5mmol/L,对碘酚0.3~0.8mmol/L,FeCl3 9~30μmol/L,二氧六烷1~5mL/L,Tris-HCl缓冲液5mmol/L,pH 8.6,避光保存;所述发光液B为浓度为1~10mmol/L的过氧化脲溶液;所述洗涤剂中含有0.3~0.5mol/L磷酸氢二钠,0.04~0.06mol/L磷酸二氢钠,2.5~3.5mol/L氯化钠,8~12mL/L Tween-20,0.1~0.3mL/L Proclin300;
所述CD47标准品梯度溶液有10个浓度的溶液,以如下方法制得:按照3~5wt%牛血清白蛋白、0.85wt%氯化钠、3~5wt%海藻糖、pH7.3~7.5的1.0mmol/L Na2HPO4-NaH2PO4的缓冲液、0.1~0.3mL/L Proclin300配制标准品稀释液,再用标准品稀释液制备400、200、50、25、5、1、0.5、0.1、0.05、0ng/mL 10个浓度梯度的CD47标准品溶液,其中0ng/mL为标准品稀释液;
所述的检测CD47的管式磁微粒化学发光免疫定量试剂盒的制备方法,包括步骤如下:
S1:磁微粒-免疫球蛋白结合分子-CD47抗体复合物的制备
将400~500μg羧基磁微粒置于在50mM、pH为5的MES缓冲液中,然后添加100~120μg的EDC和100~120μg Sulfo-NHS后,室温震动孵育40~45min,离心洗涤后,加80~120μg免疫球蛋白结合分子孵育3~3.5h,再离心洗涤,加入80~120μg CD47单克隆抗体与磁微粒-免疫球蛋白结合分子孵育3~3.5h,得到磁微粒-免疫球蛋白结合分子-CD47抗体复合物;
S2:CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的制备
1)CD47抗体-中性亲和素的制备
将15~60mg中性亲和素溶解在100mL、0.1mol/L磷酸盐缓冲液中,加入0.5~0.8mL浓度为0.2mg/mL的 CD47抗体溶液,再逐滴加50mL浓度为0.1~0.3%戊二醛溶液,避光搅拌16~20h,然后在0.1mol/L、pH为7.4磷酸钠缓冲液中透析除去戊二醛,透析完成后的溶液中加入等体积的丙三醇,得到CD47抗体-中性亲和素;所述戊二醛溶液由0.1mol/L、pH为6.8的磷酸钠缓冲液配制;
2)生物素标记的辣根过氧化物酶结合物的制备
取4~6mg辣根过氧化物酶溶于1mL蒸馏水中,加入体积为0.2mL、质量为4.3mg的过碘酸钠溶液,室温避光搅拌20min,用0.01mol/L、pH为4.4醋酸缓冲液进行过夜透析提纯后,加入0.1~0.5mg生物素,用0.2mol/L、pH为9.5碳酸盐缓冲液调pH到8.0~9.0,室温避光振摇2~2.5h,得到生物素标记的辣根过氧化物酶结合物;
3)CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物的结合
将CD47抗体-中性亲和素用PBS洗涤,加入用PBS稀释10~50倍体积生物素标记的辣根过氧化物酶结合物,在4℃下孵育1~2h,得到CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物;
S3:组装
将磁微粒-免疫球蛋白结合分子-CD47抗体复合物、CD47抗体-中性亲和素-生物素-辣根过氧化物酶复合物、样品稀释液、发光液A、发光液B、洗涤剂、CD47标准品梯度溶液和反应管及使用说明书组装成盒,即得到检测CD47的管式磁微粒化学发光免疫定量试剂盒,储存于2~8℃。
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WO2008031288A1 (fr) * | 2006-09-13 | 2008-03-20 | Beijing Hotgen Biotech Co., Ltd | Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne |
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CA3054915A1 (en) * | 2017-03-16 | 2018-09-20 | Universite Libre De Bruxelles | Detection, quantification and/or isolation of circulating tumor cells based on the expression of cd321 marker |
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WO2008031288A1 (fr) * | 2006-09-13 | 2008-03-20 | Beijing Hotgen Biotech Co., Ltd | Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne |
CN102998466A (zh) * | 2012-11-20 | 2013-03-27 | 博奥赛斯(天津)生物科技有限公司 | 生长激素磁微粒化学发光免疫定量检测试剂盒及其制备方法 |
CA3054915A1 (en) * | 2017-03-16 | 2018-09-20 | Universite Libre De Bruxelles | Detection, quantification and/or isolation of circulating tumor cells based on the expression of cd321 marker |
CN111122876A (zh) * | 2020-01-09 | 2020-05-08 | 上海东方肝胆外科医院 | Cd47抗体在肝癌放疗敏感性检测试剂盒中的应用 |
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