CN117388499A - 血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒 - Google Patents
血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒 Download PDFInfo
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Abstract
本发明公开了血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,涉及发光蛋白芯片技术领域。该血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,血清中抗原类蛋白的化学发光蛋白芯片方法,包括以下步骤:S1:标准品的制备和稀释,使用前1小时内溶解、稀释;S2:样品准备:血清、血浆、细胞培养上清、细菌或者组织裂解液的上清。上样前可以用SampleDiluent适当稀释;S3:Cy3标记的链霉亲和素准备:加入1.4mlSampleDiluent溶解备用;S4:从冰箱取出芯片,恢复室温后,撕掉盖膜,室温干燥1‑2小时;S5:加入100ulSampleDiluent室温封闭30分钟;S6:吸出封闭液,在1‑6we11中加入标准品100ul,7‑16we11中加入待测样品血清100u1;室温孵育1小时。
Description
技术领域
本发明涉及发光蛋白芯片技术领域,特别涉及血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒。
背景技术
蛋白质芯片是一种高通量的蛋白功能分析技术,可用于蛋白质表达谱分析,研究蛋白质与蛋白质的相互作用,甚至DNA-蛋白质、RNA-蛋白质的相互作用,筛选药物作用的蛋白靶点等。
蛋白芯片技术的研究对象是蛋白质,其原理是对固相载体进行特殊的化学处理,再将已知的蛋白分子产物固定其上(如酶、抗原、抗体、受体、配体、细胞因子等),根据这些生物分子的特性,捕获能与之特异性结合的待测蛋白(存在于血清、血浆、淋巴、间质液、尿液、渗出液、细胞溶解液、分泌液等),经洗涤、纯化,再进行确认和生化分析;它为获得重要生命信息(如未知蛋白组分、序列。体内表达水平生物学功能、与其他分子的相互调控关系、药物筛选、药物靶位的选择等)提供有力的技术支持。
现有的蛋白芯片,不具备有高通量的验证能力,因此我们提出了血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒。
发明内容
本发明的目的在于至少解决现有技术中存在的技术问题之一,提供血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,
为实现上述目的,本发明提供如下技术方案:血清中抗原类蛋白的化学发光蛋白芯片方法,包括以下步骤:
S1:标准品的制备和稀释,使用前1小时内溶解、稀释;
S2:样品准备:血清、血浆、细胞培养上清、细菌或者组织裂解液的上清。上样前可以用SampleDiluent适当稀释;
S3:Cy3标记的链霉亲和素准备:加入1.4mlSampleDiluent溶解备用;
S4:从冰箱取出芯片,恢复室温后,撕掉盖膜,室温干燥1-2小时;
S5:加入100ulSampleDiluent室温封闭30分钟;
S6:吸出封闭液,在1-6we11中加入标准品100ul,7-16we11中加入待测样品血清100u1;室温孵育1小时;
S7:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S8:抗体结合,每孔加入抗体80ul,室温孵育1小时;
S9:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S10:标记,每孔加入80ulCy3标记的链霉亲和素,避光室温孵育1小时;
S11:继续清洗,200ul的1X洗液I洗4遍,除去围栏,200u1的1X洗液I摇洗15分钟,200ul的1X洗液II摇洗5分钟;
S12:芯片干燥,1000g,离心3分钟。
血清中抗原类蛋白的化学发光蛋白芯片试剂盒,包括碳酸钠、碳酸氢钠、二喹啉甲酸、酒石酸钠溶于M氢氧化钠中、4%硫酸铜一次性标准白蛋白、牛血清白蛋白(BSA)存在于5%盐和4%叠氮化钠中。
与现有技术相比,本发明的有益效果是:
(1)、该血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,直接用粗生物样品(血清、尿、体液)进行分析,同时快速发现多个生物标记物,高通量的验证能力。
(2)、该血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,发现低丰度蛋白质测定疏水蛋白质:与“双相电泳加飞行质谱”相比,除了有相似功能外,并可增加测定疏水蛋白质,在同一***中集发现和检测为一体特异性高利用单克隆抗体芯片,可鉴定未知抗原/蛋白质,以减少测定蛋白质序列的工作量。
(3)、该血清中抗原类蛋白的化学发光蛋白芯片方法和试剂盒,可以定量利用单克隆抗体芯片,由于结合至芯片上的抗体是定量的,故可以测定抗原量,但一般飞行质谱不用于定量分析。
具体实施方式
本部分将详细描述本发明的具体实施例,本发明之较佳实施例中示出,但其不能理解为对本发明保护范围的限制。
在本发明的描述中,大于、小于、超过等理解为不包括本数,以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量或者隐含指明所指示的技术特征的先后关系。
本发明的描述中,除非另有明确的限定,设置、安装、连接等词语应做广义理解,所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本发明中的具体含义。
本发明提供一种技术方案:血清中抗原类蛋白的化学发光蛋白芯片方法,包括以下步骤:
S1:标准品的制备和稀释,使用前1小时内溶解、稀释;
S2:样品准备:血清、血浆、细胞培养上清、细菌或者组织裂解液的上清。上样前可以用SampleDiluent适当稀释;
S3:Cy3标记的链霉亲和素准备:加入1.4mlSampleDiluent溶解备用;
S4:从冰箱取出芯片,恢复室温后,撕掉盖膜,室温干燥1-2小时;
S5:加入100ulSampleDiluent室温封闭30分钟;
S6:吸出封闭液,在1-6we11中加入标准品100ul,7-16we11中加入待测样品血清100u1;室温孵育1小时;
S7:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S8:抗体结合,每孔加入抗体80ul,室温孵育1小时;
S9:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S10:标记,每孔加入80ulCy3标记的链霉亲和素,避光室温孵育1小时;
S11:继续清洗,200ul的1X洗液I洗4遍,除去围栏,200u1的1X洗液I摇洗15分钟,200ul的1X洗液II摇洗5分钟;
S12:芯片干燥,1000g,离心3分钟;
使用时将待检的含有蛋白质的标本如尿液、血清、***、组织提取物等,按一定程序做好层析、电泳、色谱等前处理,然后在每个芯池里点入需要的种类。一般样品量只要2-10μL即可。
根据测定目的不同可选用不同探针结合或与其中含有的生物制剂相互作用一段时间,然后洗去未结合的或多余的物质,将样品固定一下等待检测即可。
直接检测模式是将待测蛋白用荧光素或同位素标记,结合到芯片的蛋白质就会发出特定的信号,检测时用特殊的芯片扫描仪扫描和相应的计算机软件进行数据分析,或将芯片放射显影后再选用相应的软件进行数据分析。间接检测模式类似于ELISA方法,标记第二抗体分子。以上两种检测模式均基于阵列为基础的芯片检测技术。该法操作简单、成本低廉,可以在单一测量时间内完成多次重复性测量。
蛋白芯片主要有三类:蛋白质微阵列、微孔板蛋白质芯片、三维凝胶块芯片。
蛋白质微阵列
哈佛大学的Macbeath和SchreiberL等报道了:通过点样机械装置制作蛋白质芯片的研究,将针尖浸入装有纯化的蛋白质溶液的微孔中,然后移至载玻片上,在载玻片表面点上1nl的溶液,然后机械手重复操作,点不同的蛋白质。利用此装置大约固定了10,000种蛋白质,并用其研究蛋白质与蛋白质间,蛋白质与小分子间的特异性相互作用。Macbeath和Schreiber首先用一层小牛血清白蛋白(BSA)修饰玻片,可以防止固定在表面上的蛋白质变性。由于赖氨酸广泛存在于蛋白质的肽链中,BSA中的赖氨酸通过活性剂与点样的蛋白质样品所含的赖氨酸发生反应,使其结合在基片表面,并且一些蛋白质的活性区域露出。这样,利用点样装置将蛋白质固定在t3SA表面上,制作成蛋白质微阵列。
微孔板蛋白芯片
Mendoza等在传统微滴定板的基础上,利用机械手在96孔的每一个孔的平底上点样成同样的四组蛋白质,每组36个点(4×36阵列),含有8种不同抗原和标记蛋白。可直接使用与之配套的全自动免疫分析仪,测定结果。适合蛋白质的大规模、多种类的筛选。
三维凝胶块芯片
三维凝胶块芯片是美国阿贡国家实验室和俄罗斯科学院恩格尔哈得分子生物学研究所开发的一种芯片技术。三维凝胶块芯片实质上是在基片上点布以10000个微小聚苯烯酰胺凝胶块,每个凝胶块可用于靶DNA、RNA和蛋白质的分析。这种芯片可用于筛选抗原抗体、酶动力学反应的研究。该***的优点是:凝胶条的三维化能加进更多的已知样品,提高检测的灵敏度;蛋白质能够以天然状态分析,可以进行免疫测定、受体、配体研究和蛋白质组分分析。
血清中抗原类蛋白的化学发光蛋白芯片试剂盒,包括碳酸钠、碳酸氢钠、二喹啉甲酸、酒石酸钠溶于M氢氧化钠中、4%硫酸铜一次性标准白蛋白、牛血清白蛋白(BSA)存在于5%盐和4%叠氮化钠中。
储存:以上试剂保持在室温下储存和装运。
直接用粗生物样品(血清、尿、体液)进行分析,同时快速发现多个生物标记物,高通量的验证能力。发现低丰度蛋白质测定疏水蛋白质:与“双相电泳加飞行质谱”相比,除了有相似功能外,并可增加测定疏水蛋白质,在同一***中集发现和检测为一体特异性高利用单克隆抗体芯片,可鉴定未知抗原/蛋白质,以减少测定蛋白质序列的工作量,可以定量利用单克隆抗体芯片,由于结合至芯片上的抗体是定量的,故可以测定抗原量,但一般飞行质谱不用于定量分析。
上面对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所述技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。
Claims (2)
1.血清中抗原类蛋白的化学发光蛋白芯片方法,其特征在于,包括以下步骤:
S1:标准品的制备和稀释,使用前1小时内溶解、稀释;
S2:样品准备:血清、血浆、细胞培养上清、细菌或者组织裂解液的上清,
上样前可以用SampleDiluent适当稀释;
S3:Cy3标记的链霉亲和素准备:加入1.4mlSampleDiluent溶解备用;
S4:从冰箱取出芯片,恢复室温后,撕掉盖膜,室温干燥1-2小时;
S5:加入100ulSampleDiluent室温封闭30分钟;
S6:吸出封闭液,在1-6we11中加入标准品100ul,7-16we11中加入待测样品血清100u1;室温孵育1小时;
S7:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S8:抗体结合,每孔加入抗体80ul,室温孵育1小时;
S9:芯片清洗,200ul的1X洗液I洗5遍,200ul的1X洗液II洗2遍;
S10:标记,每孔加入80ulCy3标记的链霉亲和素,避光室温孵育1小时;
S11:继续清洗,200ul的1X洗液I洗4遍,除去围栏,200u1的1X洗液I摇洗15分钟,200ul的1X洗液II摇洗5分钟;
S12:芯片干燥,1000g,离心3分钟。
2.血清中抗原类蛋白的化学发光蛋白芯片试剂盒,其特征在于,包括碳酸钠、碳酸氢钠、二喹啉甲酸、酒石酸钠溶于M氢氧化钠中、4%硫酸铜一次性标准白蛋白、牛血清白蛋白(BSA)存在于 5%盐和 4%叠氮化钠中。
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