The Helix-II epitope: a cautionary tale from a cartilage biomarker based on an invalid collagen sequence

Abstract

Summary Objective An apparent database error in the sequence underlying the Helix-II cartilage biomarker immunoassay was investigated at the protein level. Methods and Results Tandem mass spectrometry established the peptide sequence ERGETGPP*GPA in human type II collagen, not ERGETGPP*GTS used to generate the antibody for the Helix-II assay. Conclusions Recent reports in which the Helix-II assay was applied to urine or serum as a marker of cartilage collagen degradation need to be re-evaluated since the epitope does not occur in cartilage type II collagen. Based on collagen sequences and Helix-II epitope properties, type III collagen is one of several candidate sources of the cross-reacting signal in body fluids, but not type II collagen. The findings highlight the need for more stringent scrutiny of the origins and validation of molecular markers in body fluid assays in general.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis

“…This information can be difficult to generate because the concentrations of the analytes found in serum and/or urine are usually very low and their determination requires complex analysis. This aspect of the biomarker validation process is however of critical importance for correct interpretation of biomarker results [41]. …”

Serum and urinary biochemical markers for knee and hip-osteoarthritis: a systematic review applying the consensus BIPED criteria

“…Note that Eyre et al recently reported their doubts about the origin of HELIX-II epitopes. They showed that the HELIX-II epitope is not present on collagen type II, but suggest its presence on collagen type III degradation products instead 15 .…”

Associations of CTX-II with biochemical markers of bone turnover raise questions on its tissue origin: data from CHECK, a cohort study of early osteoarthritis

“…A drawback of this positive drive is the limited validation and qualification of biomarkers before their introduction in research practice. For example, HELIX-II (type II helical collagen epitope) was initially introduced as a new biomarker of collagen type II degradation in humans,1 but later appeared to be erroneously raised against a peptide sequence not present in human cartilage as confirmed by mass spectrometry 2. Another example is COMP (cartilage oligomeric matrix protein), a biomarker that was presented as a biomarker specific for cartilage,3 but which is present in other tissues as well 4 5…”