Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Laura Moonen, Jules L. Derks, Lisa M.V. Lap, Britney J.C.A. Marijnissen, Lisa M. Hillen, Michael A. den Bakker, Jan H. von der Thüsen, Robert Jan van Suylen, Wim Timens, Maria Bintanel, Eugenia Kuteeva, Anne Marie C. Dingemans, Ernst Jan M. Speel*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Background: Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics. 

Methods: OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA). 

Results: Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako. 

Conclusions: New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics.

Original languageEnglish
Pages (from-to)2181-2191
Number of pages11
JournalTranslational Lung Cancer Research
Volume11
Issue number11
DOIs
Publication statusPublished - 29 Nov 2022

Bibliographical note

Funding Information:
The data of the present manuscript have been presented at the Annual ENETS conference 2022. ENETS has published all submitted abstracts, including our abstract, in a special issue in the Journal of Neuroendocrinology. Funding: This work was supported by the Dutch Cancer Foundation (grant No. 10956, 2017).

Publisher Copyright: © Translational Lung Cancer Research. All rights reserved.

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