Development and characterization of an ETV1 rabbit monoclonal antibody for the immunohistochemical detection of ETV1 expression in cancer tissue specimens

作者：Cara C. Schafer, Denise Young, Harpreet Singh, Rahul Jayakrishnan, Sreedatta Banerjee, Yingjie Song, Albert Dobi, György Petrovics, Sudhir Srivastava, Shiv Srivastava, Isabell A. Sesterhenn, Gregory T. Chesnut, Shyh‐Han Tan · 发表于：Journal of Immunological Methods · 年份：2023 · DOI：10.1016/j.jim.2023.113493 · 被引用次数：4 · 研究领域：Radiopharmaceutical Chemistry and Applications、Prostate Cancer Treatment and Research、Wnt/β-catenin signaling in development and cancer

BACKGROUND: Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function. METHODS: An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens. RESULTS: Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells. CONCLUSIONS: The sel...