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Development of a cartilage oligomeric matrix protein neo-epitope assay for the detection of intra-thecal tendon disease

Smith, Roger LU ; Önnerfjord, Patrik LU orcid ; Holmgren, Kristin LU ; Di Grado, Shacko and Dudhia, Jayesh (2020) In International Journal of Molecular Sciences 21(6).
Abstract

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses... (More)

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cartilage oligomeric matrix protein (COMP), Neoepitope, Tendinopathy, Tendon
in
International Journal of Molecular Sciences
volume
21
issue
6
article number
2155
publisher
MDPI AG
external identifiers
  • pmid:32245107
  • scopus:85082333879
ISSN
1661-6596
DOI
10.3390/ijms21062155
language
English
LU publication?
yes
id
99c6035e-782a-4109-af7c-ce425f02ef9b
date added to LUP
2020-04-07 08:56:56
date last changed
2025-01-10 10:05:25
@article{99c6035e-782a-4109-af7c-ce425f02ef9b,
  abstract     = {{<p>The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH<sub>2</sub>-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant &gt; 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.</p>}},
  author       = {{Smith, Roger and Önnerfjord, Patrik and Holmgren, Kristin and Di Grado, Shacko and Dudhia, Jayesh}},
  issn         = {{1661-6596}},
  keywords     = {{Cartilage oligomeric matrix protein (COMP); Neoepitope; Tendinopathy; Tendon}},
  language     = {{eng}},
  number       = {{6}},
  publisher    = {{MDPI AG}},
  series       = {{International Journal of Molecular Sciences}},
  title        = {{Development of a cartilage oligomeric matrix protein neo-epitope assay for the detection of intra-thecal tendon disease}},
  url          = {{http://dx.doi.org/10.3390/ijms21062155}},
  doi          = {{10.3390/ijms21062155}},
  volume       = {{21}},
  year         = {{2020}},
}