Clone e1l3n

Manufactured by Cell Signaling Technology

Sourced in United States

Clone E1L3N is a monoclonal antibody that recognizes the endogenous levels of ERK1/2 (p44/42 MAPK) protein. It can be used for Western blotting and immunoprecipitation applications.

Automatically generated - may contain errors

Lab products found in correlation

Bond 3, by Leica (4 mentions) Clone c8 144b, by Agilent Technologies (4 mentions) Clone d7u8c, by Cell Signaling Technology (2 mentions) Clone sp7, by Agilent Technologies (2 mentions) Ultraview universal dab detection kit, by Roche (2 mentions) Bond polymer refine detection kit, by Leica (2 mentions) Clone 28 8, by Abcam (2 mentions) E1l3n, by Agilent Technologies (1 mentions) Sp263 assay, by Roche (1 mentions) Clone d1m8i, by Cell Signaling Technology (1 mentions) Goat anti foxa3, by Santa Cruz Biotechnology (1 mentions) Anti cd3, by Agilent Technologies (1 mentions) Mach2 system, by Biocare Medical (1 mentions) Scn400 slide scanner, by Leica (1 mentions) Ventana discovery xt, by Roche (1 mentions) Clone nat105, by Abcam (1 mentions) Halo image analysis software, by Indica Labs (1 mentions) Benchmark ultra, by Roche (1 mentions) Clone mrq 22, by Cell Marque (1 mentions) Mini trans blot cell system, by Bio-Rad (1 mentions)

Close spelling variants of this product

E1L3N (126 citations) PD-L1 (clone E1L3N, (36 citations) Anti-PD-L1 (clone E1L3N, (10 citations) Anti-PD-L1 antibody (clone E1L3N; (4 citations) Rabbit anti-PD-L1 monoclonal antibody (clone E1L3N, (3 citations) Rabbit monoclonal antibodies for PD-L1 (clone E1L3N®) (2 citations) Anti-PD-L1 monoclonal antibody (clone E1L3N; (2 citations)

32 protocols using clone e1l3n

Comprehensive PD-L1 Immunohistochemistry Assay Evaluation

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A first set of three antibodies was tested: Clone E1L3N (Cell Signaling Technology, Danvers, MA, USA), clone 22C3 (Pharm Dx kit, DAKO, Agilent Technology, Santa Clara, CA, USA) and clone SP142 (Spring Bioscience, Pleasanton, CA, USA). We completed the study with the SP263 assay (Ventana Medical System, Tucson, USA) when it became commercially available, but we could only determine the SP263 status for 83 samples. SP142 was tested both as CA on Benchmark Ultra or as LDT on Dako autostainer, E1L3N was tested as LDT on Dako autostainer. PD-L1 22C3 PharmDx assay was performed on Dako Autostainer 48 according to the manufacturer’s instructions. PD-L1 SP263 commercial assay was performed on Benchmark Ultra according to the manufacturer’s instructions. See Table 2 for further details.

Table 2

Antibodies and technical data

Antibody	Clone	Provider	Visualisation system	Dilution
PD-L1	E1L3N (LDT)	Cell Signaling Technology	Envision Flex Sytem Dako	1/500
PD-L1	22C3 (CA)	Agilent (Dako)	Envision Flex Sytem Dako	Prediluted
PD-L1	SP263 (CA)	Roche Ventana	Optiview system Ventana	Prediluted
PD-L1	SP142 (LDT)	Roche (Spring biosciences)	Envision Flex Sytem Dako	1/100
PD-L1	SP142 (CA)	Roche (Spring biosciences)	Optiview system Ventana	1/60
PD1	Nat105	Roche Ventana	Optiview system Ventana	Prediluted
CD8	SP57	Roche Ventana	Optiview system Ventana	Prediluted
PD-L2	D7U8C	Cell Signaling Technology	Envision Flex Sytem Dako	1/100

Rouquette I., Taranchon-Clermont E., Gilhodes J., Bluthgen M.V., Perallon R., Chalabreysse L., De Muret A., Hofman V., Marx A., Parrens M., Secq V., Thomas de Montpreville V., Galateau-Salle F., Brousset P., Milia J., Girard N., Besse B., Molina T.J, & Mazières J. (2019). Immune biomarkers in thymic epithelial tumors: expression patterns, prognostic value and comparison of diagnostic tests for PD-L1. Biomarker Research, 7, 28.

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Comprehensive Lung Tissue Staining Protocol

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Staining (H&E, Alcian blue and immunohistochemistry) was performed using 5‐μm paraffin‐embedded lung sections as previously described (Chen et al, 2009) except EDTA antigen retrieval was performed for PD‐L1 staining. Antibodies used were rabbit anti‐VTCN1 (1:100; clone D1M8I; cat# 14572, Cell Signaling Technology, Danvers, MA; validated by immunoblotting; see Appendix Fig S3), rabbit anti‐PD‐L1 (1:100; clone E1L3N; cat# 13684, Cell Signaling Technology), goat anti‐FOXA3 (1:100; cat# sc‐5361, Santa Cruz Biotechnology), mouse anti‐NKX2‐1/TTF‐1 (1:500; cat# WRAB‐1231; Seven Hills Bioreagents, Cincinnati, OH), and guinea pig anti‐SPDEF (1:500; a gift from J. Whitsett; Park et al, 2007). Blind assessment of staining was performed by three investigators (K.T., I.M.F‐B., and Y.M.).

Guo M., Tomoshige K., Meister M., Muley T., Fukazawa T., Tsuchiya T., Karns R., Warth A., Fink‐Baldauf I.M., Nagayasu T., Naomoto Y., Xu Y., Mall M.A, & Maeda Y. (2017). Gene signature driving invasive mucinous adenocarcinoma of the lung. EMBO Molecular Medicine, 9(4), 462-481.

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Dual Immunohistochemistry Evaluation of PD-L1/PD-L2 and PAX5

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Double staining of PD-L1 (1:50; clone E1L3N; Cell Signaling, Danvers,
MA) or PD-L2 (1:50; clone D7U8C; Cell Signaling) and PAX5 (1:100; clone 24; BD
Biosciences, San Jose, CA) was performed with an automated staining system (Bond
III; Leica Biosystems, Buffalo Grove, IL) as previously described (Ansell et al., 2015 (link)). Stained slides were
scored by an expert dermatopathologist (JG) and expert hematopathologist (AB),
and percentages of both tumor PDL-1 or PDL-2 and microenvironment PDL-1 or PDL-2
were calculated by scoring 100–200 cells in each category. The threshold
for PD-L1 and PD-L2 expression was defined at 30% for PAX5-positive tumor cells
and at 20% for PAX5-negative immune cell microenvironment, as reported by others
(Kiyasu et al., 2015 (link)).

Zhou X.A., Louissaint A J.r., Wenzel A., Yang J., Martinez-Escala M.E., Moy A.P., Morgan E.A., Paxton C.N., Hong B., Andersen E.F., Guitart J., Behdad A., Cerroni L., Weinstock D.M, & Choi J. (2018). GENOMIC ANALYSES IDENTIFY RECURRENT ALTERATIONS IN IMMUNE EVASION GENES IN DIFFUSE LARGE B CELL LYMPHOMA, LEG TYPE. The Journal of investigative dermatology, 138(11), 2365-2376.

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Quantifying Tumor-Infiltrating CD3+ Cells

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Tumors were collected, formalin fixed and paraffin embedded. Immunohistochemistry was performed on 3 µm paraffin sections of each tumor using anti-PD-L1 (10 µg/mL, clone E1L3N, Cell Signaling, USA) or anti-CD3 (6 µg/mL, polyclonal, Agilent, USA) primary antibodies, followed by the Peroxidase/DAB Envision detection system (Agilent) on an automated platform (Dako Autosatiner), according to manufacturer’s instructions. The sections were counterstained with Mayer’s hematoxylin. As negative control, omission of the primary antibody was performed. CD3⁺ cells were quantified on whole tissue sections, both in intratumoral areas and peritumoral stroma, with the open source software Qupath, using automatic classification and positive detection workflows.35 (link) Results are expressed as the percentage of CD3+ cells relative to the total number of cells.

Marotte L., Simon S., Vignard V., Dupre E., Gantier M., Cruard J., Alberge J.B., Hussong M., Deleine C., Heslan J.M., Shaffer J., Beauvais T., Gaschet J., Scotet E., Fradin D., Jarry A., Nguyen T, & Labarriere N. (2020). Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes. Journal for Immunotherapy of Cancer, 8(1), e000311.

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Multicolor IHC for PD-L1 and SOX10

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Tissue was incubated overnight at 4 °C with both rabbit monoclonal antibody against PD-L1 (clone E1L3N, Cell Signaling Technology, Catalogue# 13684, 1:100 dilution) and mouse monoclonal antibody against SOX-10 (clone BC34, Biocare, dilution 1:200). (SOX10 analyses were part of a separate studies and are not discussed here further.) Antigen retrieval was performed using Citrate Buffer pH6 (Biocare Decloaking Chamber). Visualization was preformed using MACH2 system (Biocare), DAB (SOX10) and Fast Red (PD-L1) as chromogens, and counterstained with hematoxylin. The slides were scanned using a high-resolution scanner (Leica SCN400 Slide Scanner) at 20× magnification. PD-L1 was scored and expressed as a percentage of stained cells for tumor cells, peri- and intratumoral immune infiltrate (mostly mononuclear phagocytic cells), and other nontumor tissue cells.

Morales-Betanzos C.A., Lee H., Gonzalez Ericsson P.I., Balko J.M., Johnson D.B., Zimmerman L.J, & Liebler D.C. (2017). Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma. Molecular & Cellular Proteomics : MCP, 16(10), 1705-1717.

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Immunohistochemical Analysis of PD-1 and PD-L1 in Tumors

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Hematoxylin and eosin slides of each archival specimen were evaluated, and a block representing the overall tumor was chosen for TMA preparation. Three cores of 1 mm diameter per case were selected. Sections were cut 4 µm thick and placed on positively charged slides for immunostaining. Staining was performed using the Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ, USA) using commercially available antibodies against PD-1 on tissue-infiltrating lymphocytes (clone NAT105, diluted 1:100; Abcam, Cambridge, UK) and PD-L1 on tumor cell membranes (clone E1L3N, diluted 1:200; Cell Signaling Technology, Beverly, MA, USA). These assays had been previously validated using Formalin-Fixed Paraffin-Embedded cell-line controls.^{10 (link)}All TMAs were reviewed twice, based on antibody expression and compared with a positive control. Scoring was performed by an experienced uropathologist who was blinded to clinical outcomes. PD-L1 expression was evaluated based on the intensity and proportion of tumoral cells showing membranous or cytoplasmic staining, and was scored as follows: 0, negative (no immunoreactivity); 1, weak (5% to less than 25% of cells); 2, moderate (between 25 and 60% of cells); and 3, strong (more than 60% of cells). The numbers of PD-1 cytoplasmic-positive lymphocytes were assessed semiquantitatively.^{11 (link)}

Chipollini J., da Costa W.H., Werneck da Cunha I., de Almeida e Paula F., Guilherme O. Salles P., Azizi M., Spiess P.E., Abreu D, & Zequi S.D. (2019). Prognostic value of PD-L1 expression for surgically treated localized renal cell carcinoma: implications for risk stratification and adjuvant therapies. Therapeutic Advances in Urology, 11, 1756287219882600.

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PD-L1 Expression Evaluation in FFPE Samples

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Staining for PD-L1 was performed in FFPE 3 µm sections with a rabbit monoclonal antibody (clone E1L3N, 1:1000; Cell Signaling Technology) on the automatic Ventana Benchmark Ultra platform, using the OptiView Universal DAB detection kit and the OptiView Amplification kit from the same manufacturer. PD-L1 immunoexpression was evaluated semi-quantitatively for tumor epithelial and stromal immune cells, according to the immunoreactivity scoring system (IRS) described by Boger et al. [44 (link)]. PD-L1 expression in tumor epithelial cells was dichotomized as positive (detected) or negative (absent) by an immunoreactivity score (IRS) of 2. PD-L1 expression in immune cells of the TME was defined as low (1–5% of positive cells), intermediate (6–20% of positive cells) or high (>20% of positive cells).

Gullo I., Carvalho J., Martins D., Lemos D., Monteiro A.R., Ferreira M., Das K., Tan P., Oliveira C., Carneiro F, & Oliveira P. (2018). The Transcriptomic Landscape of Gastric Cancer: Insights into Epstein-Barr Virus Infected and Microsatellite Unstable Tumors. International Journal of Molecular Sciences, 19(7), 2079.

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Multiplex IHC for Immune Profiling

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IHC staining for cluster of differentiation 3 (CD3), cluster of differentiation 8 (CD8), and programmed death ligand 1 (PD-L1) was performed as previously described^{49 (link)} on consecutive sections. In brief, an automated immunostainer (Bond III, Leica Bio-systems) with anti-CD3 (Abcam Cambridge; clone SP7, 1:400, RRID: AB_443425), anti-CD8 (Dako, clone C8/144B, 1:100, RRID: AB_2075537), and anti-PD-L1 (Cell Signaling Technology, clone E1L3N, 1:400, RRID: AB_2687655) was used. CD3 and CD8 expression was determined using image analysis (Aperio Image Scope) and adjusted for core completeness. PD-L1 expression was assessed by a pathologist (S. Berezowska) as the proportion of positive tumor cells.

Wang J., Sun N., Kunzke T., Shen J., Zens P., Prade V.M., Feuchtinger A., Berezowska S, & Walch A. (2023). Spatial metabolomics identifies distinct tumor-specific and stroma-specific subtypes in patients with lung squamous cell carcinoma. NPJ Precision Oncology, 7, 114.

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Immunohistochemical Analysis of Breast Tumors

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Formalin-fixed and paraffin-embedded breast tumor blocks were selected from
primary FBC and NFBC tumors and diagnostic core biopsies with their
corresponding primary tumor surgical specimens. The tissue sections were
histochemically stained with hematoxylin-eosin (HE). Estrogen receptor,
progesterone receptor, Ki67, and HER2 immunohistochemical staining were
performed in a pathology lab using routine accredited procedures. Tissues from
FBC patients with high sIL-2R levels and TNBC were stained for PD-L1 and CD8.
The slides were deparaffinized in toluene and rehydrated in graded alcohols,
after which heat-induced epitope retrieval was performed, followed by a PD-L1
IHC protocol using clone E1L3N (rabbit; Cell Signaling Technology, Danvers, MA,
USA) and a CD8 IHC protocol using clone (C8/144B) (monoclonal mouse; Dako,
Agilent Technologies, Santa Clara, CA, USA).

Gonda K., Horita S., Maejima Y., Takenoshita S, & Shimomura K. (2021). Soluble interleukin-2 receptor as a predictive and prognostic marker for patients with familial breast cancer. Science Progress, 104(3), 00368504211039590.

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Immunohistochemical Quantification of CD47 and PD-L1 in Lymphoma PDX Models

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Tissue microarray (TMA) or FFPE slides of both lymphoma PDX models were prepared following standard histology procedure. A standard IHC staining protocol was performed in Bond Rx Automated IHC/ISH Slide Staining machine (Leica Biosystems) using commercial anti-CD47 antibody, clone EPR21794 (Abcam, ab218810) and anti-PD-L1 antibody, clone E1L3N (Cell Signaling Technology, 13684). These staining were followed by whole slide imaging using the NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantified and calculated H-Score (see below) using HALO® image analysis software (Indica Labs) with pathologists' peer review. The necrotic tumor regions and blank space in the tissue were excluded and the intensity of positive staining on tumor cells was scored at four levels, 0 (negative), 1 + (weak staining), 2 + (medium staining), 3 + (strong staining). The percentages of tumor cells at different intensity levels were evaluated with formula as:

H - S c o r e = (% a t 0) \times 0 + (% a t 1) \times 1 + (% a t 2) \times 2 + (% a t 3) \times 3 (H - S c o r e r a n g e i s 0 t o 300) .

Ke H., Zhang F., Wang J., Xiong L., An X., Tu X., Chen C., Wang Y., Mao B., Guo S., Ju C., He X., Sun R., Zhang L., O’Connor O.A, & Li Q.X. (2023). HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models. Scientific Reports, 13, 5419.

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