Evaluation of PD-L1 Expression Using Diverse Antibodies

The expression of PD-L1 was assessed with 3 different Abs and assays: clone 405.9A11 (referred to as 9A11, Cell Signalling Technology), SP142 (Ventana, Roche) [38 (link)], and 22C3 (Dako, Agilent Technologies) [39 (link)]. Antibodies against PD-1 (UMAB199, Origen), CD4 (EP204, Origen), FOXP-3 (ab20034, Abcam), CD8 (SP16, BioCare), and CD163 (NCL-CD163, Novocastra) were also used. The dilutions were 1:50 or those indicated in the instructions from each supplier. All IHC staining steps were performed on an automated IHC staining instrument (VENTANA, Roche) excluding the staining for clone 22C3 (PD-L1), which was performed with a Dako Auto Stainer, and optimization for each antibody was performed for minimum nonspecific staining by adjusting the primary antibody concentration and reagent incubation times. Negative/positive controls were established as recommended [39 (link)]. The immunohistochemistry assays for the 3 different clones of anti-PDL1 Abs were performed according to previously published methods [38 (link), 40 (link), 41 (link)]; a description of the IHC assay methodology is further described in the supplementary materials.

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Shi Y., Mi L., Lai Y., Zhao M., Jia L., Du T., Song Y, & Li X. (2023). PD-L1 immunohistochemistry assay optimization to provide more comprehensive pathological information in classic Hodgkin lymphoma. Journal of Hematopathology, 16(1), 7-16.

Publication 2023

clone 405.9A11 SP142 ab20034 Auto Stainer

Corresponding Organization : Peking University Cancer Hospital

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Variable analysis

independent variables

Different Abs and assays used to assess the expression of PD-L1:
- clone 405.9A11 (referred to as 9A11, Cell Signalling Technology)
- SP142 (Ventana, Roche)
- 22C3 (Dako, Agilent Technologies)

dependent variables

Expression of PD-L1

control variables

Antibody dilutions were 1:50 or as indicated in the instructions from each supplier.
All IHC staining steps were performed on an automated IHC staining instrument (VENTANA, Roche) excluding the staining for clone 22C3 (PD-L1), which was performed with a Dako Auto Stainer.
Optimization for each antibody was performed for minimum nonspecific staining by adjusting the primary antibody concentration and reagent incubation times.
Negative/positive controls were established as recommended [39 (link)].

controls

Negative/positive controls were established as recommended [39 (link)].

Annotations

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