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Published on: October 30, 2015
Anti-CD30 (Ber-H2) epitope requires structural elements as shown by mass spectroscopy and dual-site associated
Phillip Daniel Warren1, Margaret Helfrich Smith1
1Ventana Medical Systems, Tucson, Arizona, USA.
Abstract:
The Ber-H2 mouse monoclonal antibody has been in use for 35 years for detecting the CD-30 biomarker in a variety of lymphomas. Despite the wide use of this clone, we have not been successful in applying synthetic peptides derived from the published epitope sequence and affinity data toward the development of a new Ber-H2-based in vitro diagnostic reagent assay. We found that synthetic peptides based on the published epitope sequence do not function to inhibit antibody-binding activity, thus indicating that the sequence is not the full epitope recognized by Ber-H2. In this report, we used mass spectroscopic analysis of proteolyzed CD30 fragments capable of binding Ber-H2 to identify additional regions within the epitope that participate in binding. Using surface plasmon resonance binding kinetic analyses and immuno-histochemical peptide-inhibition assays, we also demonstrate that the epitope sequence as originally reported is missing two key elements necessary for binding the Ber-H2 antibody.
Insights
The Ber-H2 antibody's epitope was re-evaluated. New binding regions were identified, improving diagnostic assay development for CD-30 lymphomas.
Area of Science:
- Immunology
- Biochemistry
- Oncology
Background:
- The Ber-H2 monoclonal antibody is widely used for CD-30 biomarker detection in lymphomas.
- Previous attempts to develop Ber-H2-based in vitro diagnostic assays using synthetic peptides were unsuccessful.
Purpose of the Study:
- To identify the complete epitope recognized by the Ber-H2 antibody.
- To enable the development of novel Ber-H2-based diagnostic reagents.
Main Methods:
- Mass spectroscopic analysis of CD30 fragments.
- Surface plasmon resonance (SPR) binding kinetics.
- Immuno-histochemical peptide-inhibition assays.
Main Results:
- Synthetic peptides based on the published epitope sequence did not inhibit Ber-H2 binding.
- Mass spectrometry identified additional binding regions within the CD30 epitope.
- SPR and inhibition assays confirmed the published sequence is incomplete.
Conclusions:
- The Ber-H2 antibody recognizes a larger epitope than previously reported.
- The identified epitope regions are crucial for Ber-H2 binding.
- This research facilitates the development of improved diagnostic tools for CD-30-related lymphomas.
