CN105717298A - Pd-l1免疫组化试剂盒 - Google Patents

Pd-l1免疫组化试剂盒 Download PDF

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CN105717298A
CN105717298A CN201610064240.7A CN201610064240A CN105717298A CN 105717298 A CN105717298 A CN 105717298A CN 201610064240 A CN201610064240 A CN 201610064240A CN 105717298 A CN105717298 A CN 105717298A
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antibody
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徐军
李兴民
严旭霞
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Zhejiang Shuo Wen Bioisystech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

本发明涉及检测人组织特别是人肿瘤组织中PD?L1蛋白表达的免疫组化试剂和试剂盒及其制备方法和应用。

Description

PD-L1免疫组化试剂盒
技术背景
程序性死亡分子1(PD-1)主要表达于活化的T淋巴细胞、B淋巴细胞和巨噬细胞表面。程序性死亡分子1配体-1(PD-L1)，又称B7同源分子1(B7-H1)是PD-1的配体，属于I型跨膜蛋白。PD-L1与PD-1结合后，由PD-1传递抑制性信号，能够抑制淋巴细胞的增殖和活化。近几年发现，一些病人的肿瘤细胞表面高度表达PD-L1，通过与肿瘤浸润淋巴细胞表面的PD-1分子结合，抑制淋巴细胞的功能，从而导致肿瘤发生免疫逃逸。临床实验研究表明，阻断PD-1或PD-L1可以有效地治疗或抑制肿瘤生长。因此，临床上需要简便易行的检测手段筛选出肿瘤组织或细胞高度表达PD-L1的肿瘤患者，为个性化地进行阻断PD-1或PD-L1的治疗提供指导和依据。
附图简介
图1是用6E8和MS-1215组合对人胎盘组织进行免疫组化染色结果。
图2是用6E8和859643组合对人胎盘组织进行免疫组化染色结果。
图3是用6E8和ENS004组合对人胎盘组织进行免疫组化染色结果。
图4是用6E8和KIT-5010组合对人胎盘组织进行免疫组化染色结果。
图5是用RIgG和MS-1215组合对人胎盘组织进行免疫组化染色结果。
图6是用6E8和MS-1215组合对人肺鳞癌组织进行免疫组化染色结果。
图7是用图6的对人肺鳞癌组织进行免疫组化阴性对照染色结果。
发明概要
本发明涉及一种PD-L1免疫组化试剂盒，用于检测人组织中，特别是人肿瘤组织中PD-L1蛋白的表达。免疫组化试剂盒一般至少包括用于特异性结合目的标记蛋白的一抗和结合一抗的带有可以被检测出的标记的二级抗体，这两种抗体的组合配对至关重要，找到一个最佳组合是开发一个优质免疫组化产品的关键。发明人针对抗人PD-L1抗体(一抗)和二抗进行了大量的试验，筛选了无数的组合，经过了反复的失败，终于找到了几个最佳组合，进而开发出了特异性强、灵敏度高的PD-L1免疫组化试剂盒。
因此，一方面，本发明的PD-L1免疫组化试剂盒至少包括(1)抗人PD-L1抗体；和(2)可以和所述抗人PD-L1抗体结合的并带有可以被检测出的标记的二级抗体。其中抗人PD-L1抗体可以是鼠单抗、鼠多抗、兔单抗或兔多抗。已知的可以选用的抗人PD-L1抗体包括Abcam Anti-PD-L1antibody 28-8货号ab205921、AbcamAnti-PD-L1antibody货号ab58810、AbcamAnti-PD-L1 antibody货号ab153991、AbcamAnti-PD-L1抗体MIH2货号ab109052、Thermo Fisher PD-L1/CD274Antibody货号PA5-20343、Thermo Fisher PD-L1/CD274 Antibody货号PA5-30937、Thermo Fisher PD-L1/CD274 Antibody货号PA5-28115、AbDSerotec Rabbit anti Human CD274货号AHP1703、AbDSerotec Goat anti Human CD274(C-Terminal)货号AHP2128、MicroSpin(微旋)Anti-PD-L1 6E8(货号MS-1028)、Spring bioscience PD-L1/CD274 SP142货号M4420、Cell Signaling TechnologyPD-L1Rabbit mAb货号13684、Cell Signaling Technology PD-L1(405.9A11)Mouse mAb货号29122、Dako 28-8以及Dako 22C3。
可以和所述抗人PD-L1抗体结合的并带有可以被检测出的标记的二级抗体可以选自：深圳欣博盛酶标二抗货号ENS004、福州迈新酶标二抗货号KIT-0015、福州迈新酶标二抗货号KIT-0016、福州迈新酶标二抗货号KIT-5010、福州迈新酶标二抗货号KIT-9921、MicroSpin(微旋)酶标二抗货号MS-1215、Life Technologies酶标二抗货号859643、中杉金桥酶标二抗货号ZB-2301、eBioscience酶标二抗货号18-4818、Abcam酶标二抗货号ab20043、ThermoFisher酶标二抗货号A16110、Sigma-Aldrich酶标二抗货号A0168、以及Sigma-Aldrich酶标二抗货号SAB3701066。
可选地，试剂盒可以还包含至少一种酶标底物作为显色剂例如显红色的AEC(3-氨基-9-乙基咔唑(3-amino-9-ethyl-carbazole)或显棕黄色的DAB(二氨基联苯氨Diaminobenzidin,DAB)。DAB可以是广州深达DAB货号KS003、博士德DAB货号AR1000、博士德DAB货号AR1026、中杉金桥DAB货号ZLI-9017、中杉金桥DAB货号ZLI-9031、迈新DAB货号DAB-0031、迈新DAB货号DAB-2031、SantaCruzBio DAB货号sc-24982、或者Abcam DAB货号ab103723。
试剂盒还可以包含复染剂用于将细胞核染色以彰显细胞轮廓。适用的复染剂可以是苏木素(hematoxylin，染色后呈蓝色)、甲基绿(methyl green,染色后呈绿色)、或核快红(nuclearfast red，呈红色)。
合适的显色剂+复染剂组合可以是下列任一种：DAB+苏木素；DAB+甲基绿；DAB+核快红；AEC+苏木素；或AEC+甲基绿。
在一个实施方案中，抗人PD-L1抗体(一抗)是Dako28-8。
在一个实施方案中，抗人PD-L1抗体(一抗)是Dako22C3。
在一个实施方案中，抗人PD-L1抗体(一抗)是6E8(MS-1028)。
在一个实施方案中，二抗是羊抗鼠/兔二抗MS-1215。
在一个实施方案中，二抗是羊抗鼠/兔二抗Life Technologies货号859643。
在一个实施方案中，二抗是Abcam酶标二抗货号ab20043。
在一个实施方案中，二抗是Thermo Fisher酶标二抗货号A16110。
在一个实施方案中，二抗是Sigma-Aldrich酶标二抗货号A0168。
在一个实施方案中，一抗选自6E8、28-8、和22C3；二抗选自羊抗鼠/兔二抗MS-1215和羊抗鼠/兔二抗859643；显色剂是DAB；复染剂是苏木素。
在应用本发明的试剂盒时，使用抗体和目标抗原位点(PD-L1)结合，再与标记过的二抗进行反应，最后通过显色反应呈现组织切片中的目标抗原位点，通过光学显微镜可确定目标抗原位点的分布和含量，其结果不仅能作为临床医生的诊断依据，而且可以为PD-L1抑制剂/抗体药物筛选临床适用人群并提供临床用药指导。
实施例
对比不同试剂厂家生产的二抗，优选最佳配对方案。
免疫组化实验步骤：
1)脱蜡：置于60℃烤箱预热30min，然后分别放入二甲苯I&II中各15分钟；
2)水化：无水乙醇I&II中各5分钟，95％、85％、75％乙醇各2分钟；
3)内源性过氧化物酶灭活：滴加3％H2O2，室温静置5分钟；
4)PBS洗3次，每次3分钟；
5)封闭：滴加封闭液，37℃孵育30分钟，除去多余液体；
6)一抗：加入适当浓度的一抗覆盖组织，室温孵育1小时
7)PBS洗3次，每次3分钟；
8)二抗：加入酶标二抗覆盖组织，室温孵育30分钟；
9)PBS洗3次，每次3分钟；
10)显色：加入配制好的DAB显色液，室温静置1-5分钟，观察颜色适时终止；
11)自来水冲洗3分钟；
12)复染：加入苏木素，室温静置5-60秒，自来水冲洗；
13)脱水：75％、85％、95％以及无水乙醇I&II中各2分钟；
14)透明：二甲苯I&II中各2分钟；
15)使用适量封片剂封片，显微镜下观察并拍照。
一抗选用6E8(MS-1028)、28-8、23C3，二抗选用微旋(MicroSpin)MS-1215、Life technologies(859643)、欣博盛(ENS004)、迈新(KIT-0015)。图1是用6E8和MS-1215组合对人胎盘组织进行免疫组化染色结果。图2是用6E8和859643组合对人胎盘组织进行免疫组化染色结果。图3是用6E8和ENS004组合对人胎盘组织进行免疫组化染色结果。图4是用6E8和KIT-5010组合对人胎盘组织进行免疫组化染色结果。图5是用RIgG和MS-1215组合对人胎盘组织进行免疫组化染色结果。图6是用6E8和MS-1215组合对人肺鳞癌组织进行免疫组化染色结果。图7是用图6的对人肺鳞癌组织进行免疫组化阴性对照染色结果。
结果如图显示，MS-1215和Life Technologies(859643)效果明显优于其它二抗：细胞膜染色最明显，背景最低，细胞结构显示最清晰。
该实验结果应清晰明了，配对方案一(一抗：6E8；二抗：MS-1215或Life Technologies(859643))，而其余配对方案实验结果不明确，背景有较重非特异性染色，或者染色模糊、不清晰、甚至没有染色，因此配对方案一为最佳配对方案。

Claims (2)

1.一种免疫组化试剂盒包括：
(1)抗人PD-L1抗体，选自MS-1028(6E8)、ab205921(28-8)或22C3；
(2)可以和所述抗人PD-L1抗体结合的并带有可以被检测出的标记的二级抗体，选自羊抗鼠/兔二抗MS-1215和羊抗鼠/兔二抗859643；以及
(3)显色剂。
2.权利要求1所述试剂盒，其中抗人PD-L1抗体是6E8。
CN201610064240.7A 2016-01-29 2016-01-29 Pd-l1免疫组化试剂盒 Pending CN105717298A (zh)

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