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

Cara Schafer, Denise Young, Harpreet Singh, Rahul Jayakrishnan, Sreedatta Banerjee, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Sudhir Srivastava, Shiv Srivastava, Isabell A. Sesterhenn, Gregory T. Chesnut, Shyh Han Tan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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 selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

Original languageEnglish
Article number113493
JournalJournal of Immunological Methods
Volume518
DOIs
StatePublished - Jul 2023
Externally publishedYes

Keywords

  • Antibody characterization
  • ETV1
  • Prostate cancer
  • Rabbit monoclonal antibody
  • cancer diagnosis

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