克隆28-8、SP142、73-10 实验数据对比 除28-8外,Abcam还开发了针对PD-L1的兔单抗EPR19759,该抗体在IP和WB等应用中表现十分优异。
Product Name |
Clone 28-8 |
Clone 73-10 |
Clone SP142 |
Clone EPR20529 |
Antibody type |
Recombinant |
Recombinant |
Recombinant |
Recombinant |
Epitope |
Extracellular |
Intracellular |
|
|
Host/Clonality |
Rabbit monoclonal |
Rabbit monoclonal |
Rabbit monoclonal |
Rabbit monoclonal |
Species reactivity |
Human |
Human |
Human |
Mouse |
Applications verified |
IHC-P, ICC/IF, Flow cyt, WB |
IHC-P, WB, ICC/IF, WB, Flow cyt |
IHC-P, ICC/IF, IP, WB |
ICC/IF, IP, WB |
Ideal usage |
IHC Autostainer |
IHC Autostainer |
ICC Human |
ICC Mouse |
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of lung cancer tissue samples. Comparing the staining PD-L1 with different monoclonal antibodies. ab228415 (73-10) 显示出比其他PD-L1克隆号更高的灵敏度。
PD-L1 IHC 打分机制 使用不同的诊断抗体,针对不同的肿瘤类型均有其独有的PD-L1打分机制。Opdivo 在 NSCLC 的临床诊断中使用了 PD-L1 兔单抗 28-8,它只统计肿瘤细胞膜染色(TC)的百分比。而 SP142 时,除了TC 之外,也加入了对于肿瘤浸润的免疫细胞(IC)染色的评判,是仅有一个对肿瘤细胞和肿瘤浸润的淋巴细胞均加以统计的检测抗体:评估基于PD-L1任何强度表达的肿瘤浸润性免疫细胞占肿瘤面积的比例(% IC)或 PD-L1 任何强度表达在肿瘤细胞中的比例(% TC)。然而这两种打分机制并无优劣之分。
图6: PD-L1 28-8 的打分规则[5] PD-L1 IHC 28-8 pharmDx 打分规则:如图6所示,PD-L1 蛋白表达的定义是肿瘤细胞所呈现的任何强度的细胞膜阳性的百分率,细胞质染色(如果存在)不参与评分。根据阳性参照(可使用细胞石蜡切片)的染色结果将染色强度分为 0(negative)、1+(weak intensity)、2+(moderate intensity)以及 3+(strong intensity);在至少两个染色强度条件下,至少 80% 细胞显示膜染色阳性,负参照背景强度小于1同时无膜着色信号。
不完整的肿瘤细胞膜染色 pattern 和完整的肿瘤细胞膜染色 pattern 均在打分范围内;
在整个样本中不仅膜染色,也出现细胞质阳性染色,应排除在打分之外;
免疫细胞染色,应排除在打分之外(图6)[5] 。也可以参考表5中的一些建议尝试解决自动化检测平台中的常见问题。
Problem |
Probable Cause |
Suggested Action |
1. No staining of slides. |
1a. Programming error. |
1a. Verify that the SK005 PD-L1 IHC 28-8 pharmDx protocol was selected for programming of slides. |
1b. Lack of reaction with DAB+ Substrate-Chromogen Solution. |
1b. Verify that DAB+ Substrate-Chromogen Solution was prepared properly. |
1c. Sodium azide in wash buffer. |
1c. Use only Dako Wash Buffer, Code K8007. |
1d. Degradation of Control Slide. |
1d. Check kit expiration date and kit storage conditions on outside of package. |
2a. Weak staining of specimen slides. |
2a. Inappropriate fixation method used. |
2a. Ensure that only approved fixatives and fixation methods are used. |
2b. Weak staining of specimen slides or of the positive cell line on the Dako-provided Control Slide. |
2b. Inadequate target retrieval. |
2b. Verify that the 3-in-1 pre-treatment procedure was correctly performed. |
3. Excessive background staining of slides. |
3a. Paraffin incompletely removed. |
3a. Verify that the 3-in-1 pre-treatment procedure was correctly performed. |
3b. Slides dried while loading onto the Autostainer Link 48. |
3b. Ensure slides remain wet with buffer while loading and prior to initiating run. |
3c. Nonspecific binding of reagents to tissue section. |
3c. Check for proper fixation of the specimen and/or the presence of necrosis. |
4. Tissue detached from slides. |
4a. Use of incorrect microscope slides. |
4a. Use Dako FLEX IHC Microscope Slides, Code K8020, or Fisherbrand Superfrost Plus charged slides. |
4b. Inadequate preparation of specimens. |
4b. Cut sections should be placed in a 58 ± 2 °C oven for one hour prior to staining. |
5. Excessively strong specific staining. |
5a. Inappropriate fixation method used. |
5a. Ensure that only approved fixatives and fixation methods are used. |
5b. Inappropriate wash buffer used. |
5b. Use only Dako Wash Buffer, Code K8007. |
6. The Target Retrieval Solution is cloudy in appearance when heated. |
6. Components in the Target Retrieval Solution cause the reagent to appear cloudy when heated. |
6. No action required. This is normal and does not affect staining. |
表5: PD-L1 IHC 28-8 pharmDx 自动化检测平台的常见问题和解决方案 28-8 自动化及手动操作指南手工操作: SMART网站中的数据显示PD-L1是一个免疫球蛋白类似的靶标。其膜外结构域仅存在两个较小的亲水结合位点[8],其独特的免疫原 huPD-L1 (Phe19-Thr239) 设计(其他同类抗体大多为N端 1-250 AA),这也可能是 PD-L1 靶标在使用 28-8 IHC-P 检测操作条件严格的重要原因;同时在正常的或者一些类型的肿瘤样本中低表达甚至无 PD-L1 的表达;鉴于此,Abcam 实验室建议使用表4中推荐进行操作,其中独有的高温/PH 稳定的抗原修复液(建议高压121°C,2-4 min)和 polymer HRP 的显色液是染色成功的关键 (点击链接,查看protocol)。
|
备注 |
IHC 用量 |
2-7.5µg/mL |
同型对照抗体 |
Rabbit IgG, monoclonal [EPR25A] (ab172730) |
抗原修复液 |
Universal HIER antigen retrieval reagent (ab208572) |
显色系统 |
IHC detection kit HRP/DAB (ab209101) |
阳性对照 |
L2987 cell lines/CHO-PDL1(Cell Lines: Positives: B-CPAP- high, ES-2- medium, HCC70 – low) |
阴性对照 |
MCF-7 cell lines |
表4: 28-8 的实验注意事项
对于自动化免疫组仪平台和系统,推荐 BioGenex i6000 with off-line HIER、 Leica BOND RX with on-line HIER、 Ventana Ultra with on-line HIER以及 Dako Omnis 四种平台(点击链接,查看protocol)。
1)如果根据 HE 评估样本是否合适(如无肿瘤组织和形态以及是否坏死); 2)水平质控是否合格(试剂对照和组织对照);拍照时所有样本除了调整视野和焦距以外,显微镜其他部件一律不能动。 3)所有病例都必须采用配套增强试剂盒; 4)不合适的固定方法和时间会直接导致假阴性; 5)房间温度需控制在18~25℃,保持恒定的室温是染色成功的基本条件; 6)可靠的评估系统。
针对“one drug, one assay”现象,由FDA牵头,四家PD抑制剂研发企业和两家学术机构参与的“蓝印倡议”(Blueprint Initiative)正在试图找到其可能的一致性,希望在不久的将来,针对PD-L1 IHC的检测更加规范化。
1. Chen, D.S. and I. Mellman, Elements of cancer immunity and the cancer-immune set point. Nature, 2017. 541(7637): p. 321-330. 2. Ancevski Hunter, K., M.A. Socinski, and L.C. Villaruz, PD-L1 Testing in Guiding Patient Selection for PD-1/PD-L1 Inhibitor Therapy in Lung Cancer. Mol Diagn Ther, 2018. 22(1): p. 1-10. 3. Cogswell, J., et al., An Analytical Comparison of Dako 28-8 PharmDx Assay and an E1L3N Laboratory-Developed Test in the Immunohistochemical Detection of Programmed Death-Ligand 1. Mol Diagn Ther, 2017. 21(1): p. 85-93. 4. Hirsch, F.R., et al., PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. J Thorac Oncol, 2017. 12(2): p. 208-222. 5. Phillips, T., et al., Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer. Appl Immunohistochem Mol Morphol, 2015. 23(8): p. 541-9. 6. Velcheti, V., et al., Programmed death ligand-1 expression in non-small cell lung cancer. Lab. Invest., 2014. 94(1): p. 107-16. 7. Liao Z, Z.Y., Cai W, Zhang H, Alleman-Sposeto J, Smith S, et al., IHC performance of two rabbit anti-human PD-L1 monoclonal antibodies. Pleasanton: Spring Bioscience; 2014. https://products.springbio.com/products/PD-L1-CD274-SP142-/M442. 8. Ishida, Y., et al., Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J, 1992. 11(11): p. 3887-95. |