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. 2020 Jun 8;2(3):100082. doi: 10.1016/j.ocarto.2020.100082

Serological CTX-II does not measure the same as urinary CTX-II

Yunyun Luo a,b,∗, Yi He a, Morten Karsdal a, Anne-Christine Bay-Jensen a
PMCID: PMC9718164  PMID: 36474683

Summary

Objective

Type II collagen is the most abundant protein of articular cartilage. The urinary cross-linked C-terminal telopeptide of type II collagen (uCTX-II) is a matrix metalloproteinase (MMP) cleaved fragment and may be the most well-validated biomarker in osteoarthritis. The aim was to develop a serological immunoassay of CTX-II (sCTX-II) and evaluated both sCTX-II and uCTX-II levels in a cross-sectional osteoarthritis cohort.

Methods

The biological relevance of sCTX-II was validated in bovine cartilage explants cultured in the presence of Oncostatin M and tumor necrosis factor alpha (OT) or OT supplemented with GM6001 for 3 weeks. Serum and urine samples from an osteoarthritis cohort were assayed using sCTX-II and uCTX-II, respectively. Spearman's correlation was performed to evaluate the correlation between sCTX-II and uCTX-II. The association between the level of biomarkers and clinical variables was also investigated.

Results

The supernatant analyzed in sCTX-II showed significant higher CTX-II levels in the end phases of explant culture compared to the vehicle group. The release of CTX-II was completely suppressed by GM6001. The sCTX-II levels in serum were not associated with uCTX-II in urine although sCTX-II levels in urine were significantly correlated with uCTX-II. uCTX-II correlated with age and gender while sCTX-II did not. sCTX-II cannot demonstrate any clinical relevance in a cross-sectional OA cohort as uCTX-II did.

Conclusion

The sCTX-II assay can reflect the MMP-mediated type II collagen degradation in bovine cartilage explants. However, sCTX-II and uCTX-II assays show different patterns suggesting the presence of CTX-II in blood may be different from that of urine.

Keywords: Cartilage degradation, CTX-II, Matrix metalloprotease, Osteoarthritis, Type II collagen

1. Introduction

A hallmark of osteoarthritis (OA) is the degradation of the extracellular matrix (ECM) in articular cartilage. Type II collagen is the most abundant matrix protein of articular cartilage and is highly specific for this tissue. Numerous studies have proven that cartilage, especially type II collagen, degradation is an essential step in the progression of knee OA [1,2].

The CTX-II is a cross-linked C-terminal telopeptide of type II collagen and contains a dimeric-hexapeptide epitope (EKGPDP) with a pyridine ring as a linker, which is cleaved by matrix metalloproteinases (MMPs) [[3], [4], [5]]. CTX-II diffuses from the joint to blood and is ultimately excreted into the urine. Urinary CTX-II (uCTX-II) was originally described by D. Eyre [3] and later on, a commercial assay was launched in 2001 [6]. It has been suggested to be the most tested and best validated biochemical marker for assessing collagenolysis in articular cartilage during OA [[7], [8], [9], [10]]. uCTX-II is elevated in knee OA [11,12], hip OA [13,14] and rheumatoid arthritis (RA) [15,16] patients when compared to healthy subjects. Patients with higher baseline CTX-II levels have a greater risk of knee OA progression [12,17]. Additionally, uCTX-II is associated with knee pain [18] although pain associations have varied [19]. Furthermore, uCTX-II can predict the effectiveness of anti-inflammatory therapy in knee OA [20], even though no predictions are seen in other trials [21]. OA patients with high uCTX-II levels showed markedly decreased uCTX-II after treatment with nimesulide but not ibuprofen [20]. Although the lack of approved disease-modifying osteoarthritis drugs (DMOADs) limited the clinical use of uCTX-II, uCTX-II may be applied in early phase evaluation of the efficacy of DMOADs, OA progression and monitoring health status in the general population [22].

It has been reported that both serological CTX-II (sCTX-II) and uCTX-II levels are related to arthritis onset and cartilage destruction in rats with collagen-induced arthritis, suggesting sCTX-II are in line with uCTX-II in rats [23,24]. However, knowledge about sCTX-II levels in human is limited as no comprehensive study has been reported to date. Whether this is due to the lack of high sensitivity in the current uCTX-II assay is unclear. Considering serum may have less analytic and biologic variability than urine, it is warranted for the development of sCTX-II immunoassay in human. Thus, in this study, we aimed to develop a sCTX-II assay by using the same antibody from uCTX-II (Urine CartiLaps ELISA, IDS, UK) on a highly sensitive electrochemiluminescent platform to determine the levels of CTX-II in blood and to investigate sCTX-II as well as uCTX-II in a cross-sectional OA cohort.

2. Methods

2.1. Materials

All chemicals were bought from either Sigma-Aldrich or Merck unless otherwise stated. 96-well Gold streptavidin microtitre plate, Sulfo-TAG labeling kit, 4x read buffer T with a surfactant, and QuickPlex SQ 120 reader with Discovery Workbench software was purchased from Meso Scale Diagnostics (Gaithersburg, USA).

2.2. Assay protocol of sCTX-II ECLIA

A sandwich assay was developed using the electrochemiluminescence (ECL) technology on the Mesoscale Discovery (MSD) platform [25]. The antibody was the same as the one employed in uCTX-II ELISA. Briefly, the MSD Gold 96-well streptavidin plate was blocked with 100 μL of blocking buffer (10 mM PBS, 5% BSA, pH 7.4) and incubated for 1 h at 20 °C. After 3 times washing, the plate was coated with 3 μg/mL of the biotinylated antibody, dissolved in assay buffer (50 mM PBS, 1% BSA, 0.1% Tween-20, 8 g/L NaCl, 0.93% Titriplex® III, 5% Liquid II, pH 7.4) and incubated for 1 h at 20 °C. The plate was washed three times and 50 μL of the fetal bovine serum calibrators or samples were added to the appropriate wells. The plate was incubated overnight at 2–8 °C. Then 25 μL of 4 μg/mL Sulfo-TAG-labeled detection antibody was added into the plate followed by incubation for 1 h at 20 °C. Finally, 150 μL 2x Read buffer T was added, and the plate was read immediately on a MESO QuickPlex SQ 120 reader. All the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed three times with 10 mM PBS +0.05% Tween-20, pH 7.4.

2.3. Technical evaluation of sCTX-II ECLIA

The intra-assay and inter-assay coefficient of variation (CV) was calculated as the mean value of the variation of five samples analyzed 10 times in duplicate. The dilution recovery was assessed in three individual human serum samples which were diluted by the assay buffer in increments of 10% and measured in the sCTX-II ECLIA. The measuring range was defined as the range between LLOQ (lower limit of quantification) and ULOD (upper limit of detection). The concentrations determined for the diluted samples corresponded well with the back-calculated values. Similarly, it was assessed as to whether the serum could be spiked into the serum. Three human serum samples were diluted in increments of 20% in assay buffer before spiked with another human serum in the ratio of 1:1, and the measured concentrations expressed as a percentage of the expected values.

2.4. Biological validation of sCTX-II ECLIA in bovine cartilage explants model

The bovine articular cartilage explants model were set up as previously described [26]. Briefly, full depth cartilage explants were isolated from the medial femoral condyle of cattle aged 1–2 years bought from the local slaughter (Harald Hansens Slagter, Slangerup, Denmark). The cartilage explants were incubated in DMEM/F-12 (Life Technologies, US) medium with 1% penicillin and streptavidin in 96-well plate at 37 °C, 5% CO2. There were six replicates for each treatment: 1) medium alone (WO); 2) catabolic stimulation with 10 ng/mL Oncostatin M and 20 ng/mL tumor necrosis factor alpha (TNF-α) (OT); 3) OT supplemented with 10 μM GM6001 (OT+GM6001). The model was cultured for 21 days. The conditioned medium was changed every two or three days, and the supernatant was stored at −20 °C until use.

2.5. Clinical evaluation of sCTX-II and uCTX-II assays in OA cohort

As reported previously [27,28], the C4Pain cohort consisted of 281 patients with different intensity of knee joint pain. The maximal pain during the last 24 h was rated on a 10-cm continuous 0 to 100 visual analog scale (VAS) (0: no pain, 100: maximum pain). Since some samples were run out, only 245 patients were included in the present study (Fig. 1). Among the 245 participants, 188 had a diagnosis of primary knee OA according to the American College of Rheumatology (ACR) criteria [29], while 57 subjects with Kellgren-Lawrence (KL) grades ≤1 was defined as Non-OA control. Blood and urine were collected upon overnight fasting prior to surgery or during the consultation. All participants provided informed consent prior to enrollment, according to the Declaration of Helsinki. The study was approved by The Ethical Committee of Northern Jutland (VEK no.: N-20100094).

Fig. 1.

Fig. 1

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Patient flow diagram of C4Pain cohort. From a total sample of 281 individuals, 245 subjects were selected. The basis for selection included availability of biospecimens, radiographic and clinical data.

Biomarkers of sCTX-II and uCTX-II were analyzed in both serum and urine. uCTX-II was measured using the Urine CartiLaps (IDS, UK), which is a competitive enzyme-linked immunosorbent assay (ELISA) [6]. The analyses were performed according to the manufacturer's instructions. Briefly, biotinylated, synthetic CTX-II peptides are bound to the surface of streptavidin-coated wells of the microtitre plate. After washing, standards, controls, and urine samples are pipetted into the wells followed by addition of a solution of the monoclonal antibody. The wells are washed, and a solution of peroxidase-conjugated rabbit anti-mouse immunoglobulin is added to the wells. Following the second washing, tetramethylbenzidine (TMB) is added to all wells and the colour reaction is stopped with 0.18 M sulfuric acid and the absorbance is measured. The concentration of uCTX-II and sCTX-II in urine was normalized to the urine creatinine by using the QuantiChrom Creatinine Assay Kit (BioAssay Systems, US). Samples falling above the ULOD were re-assayed at greater dilutions. Undiluted samples falling below LLOQ were imputed by being assigned half the LLOQ as its measured concentration.

2.6. Statistical analysis

Results are presented as the mean ± SEM. GraphPad Prism software (version 7.01) was used for all the statistical analysis except for the adjustment of body mass index (BMI), sex and age by MedCal (version 15). Differences between mean values were compared by the non-parametric Kruskal-Wallis test. The association between the level of sCTX-II and uCTX-II, demographic variables were analyzed by non-parametric Spearman's correlation. Patient tertiles of equal size depending on the sCTX-II and uCTX-II levels were made, and the difference in clinical parameters was investigated.

3. Results

3.1. Technical performance of sCTX-II ECLIA

The characterization of the antibody used in sCTX-II ECLIA was described before [6,23]. The technical performance of this ECLIA and the sCTX-II ELISA was summarized in Supplementary Table 1. The intra-assay CV was 3%, and the inter-assay CV was 11%. The measurement range was 0.005–0.68 ng/mL. The limit of detection (LOD) defined as the concentration corresponding to 3 SD below the mean of 21 determinations of the zero calibrator was 0.003 ng/mL. The mean dilution and spiking recovery tested in human serum were 92% and 106% respectively, which were within the measurement range of the assay (100 ± 20%).

3.2. Measurement of CTX-II fragments in the bovine cartilage explants model by sCTX-II ECLIA

Due to the same immunogen sequence expected in bovine, and easy access to bovine cartilage, we investigated the specificity of the sCTX-II ECLIA by using the bovine articular explants model. The explants were cultured for 21 days ex-vivo in the presence of Oncostatin M plus TNF-α (OT), which is known to induce MMP-mediated cartilage degradation. The supernatant analyzed in the sCTX-II ECLIA showed significant higher CTX-II levels in day 17 and 19 compared to the vehicle group (P < 0.001, Fig. 2). In contrast, when OT co-cultured with GM6001, which is known as a general MMPs inhibitor, the release of CTX-II was completely suppressed in comparison to the OT group (P < 0.001, Fig. 2).

Fig. 2.

Fig. 2

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Quantification of MMP-mediated type II collagen degradation products (CTX-II) in bovine explants treated with medium alone (WO), TNF-α plus oncostatin M (OT), or OT plus MMP inhibitor, GM6001 (OT + GM6001). Type II collagen degradation was quantified in the sCTX-II ECLIA. Two-way ANOVA was used to compare each group with OT. ∗∗∗∗P < 0.0001. CTX-II: crosslinked C-terminal neo-epitopes of type II collagen, ECLIA: electrochemiluminescence immunoassay, MMP: matrix metalloproteinase, TNF-α: tumor necrosis factor alpha.

3.3. Subjects characteristics

All the subjects were divided into four groups according to their KL grades and VAS scores. There was no marked difference in sex, age, BMI, race, and serum CTX-II levels across the groups except that the age in the pain group was a bit lower when compared to the healthy controls (Table 1). The pain scores (VAS and WOMAC) were significantly higher in the pain group and the radiographic OA (ROA) with pain group (P < 0.0001). The KLG in ROA group either with pain (ROA+S) or without pain (ROA-S) were significantly higher than that of the healthy controls.

Table 1.

Characteristics of C4Pain Cohort. One-way ANOVA analysis was used to compare the difference of clinical parameters in the other three groups when compared to the healthy control. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Variables Pain Control
 Healthy Control
 Radiographic OA with pain (ROA + S)
 Radiographic OA without pain (ROA-S)
n Mean SD n Mean SD n Mean SD n Mean SD
Demographic
Age (yrs) 38 59.84∗∗ 9.10 19 67.74 3.30 163 64.79 7.35 25 64.92 7.96
BMI (kg/m2) 38 26.60 3.13 19 28.10 5.00 163 28.85 4.34 25 27.37 3.29
Sex (female %) 19 50.00 11 57.89 91 55.83 13 52.00
Race (white %) 38 100 19 100 163 100 25 100
Clinical
Pain score, VAS 38 45.26∗∗∗∗ 23 19 1.21 2.39 163 53.62∗∗∗∗ 22.38 25 2.96 3.96
WOMAC pain 38 7.63∗∗∗∗ 2.78 19 1.05 2.55 163 8.04∗∗∗∗ 3.00 25 2.00 2.20
WOMAC stiffness 38 3.34∗∗∗∗ 1.58 19 0.42 0.90 163 3.84∗∗∗∗ 1.50 25 1.08 1.26
WOMAC function 38 20.24∗∗∗∗ 11.63 19 1.53 3.96 163 21.71∗∗∗∗ 11.11 25 4.80 5.81
sCTX-II (pg/mL) 38 521.8 1184 19 263.9 403.4 162 245.7 402 25 278.0 464.3
uCTX-II (ng/mmoL Creatinine) 38 395.2 237.7 19 369.2 164.5 163 526.4∗ 259.9 25 439.0 185.3
Radiographic
KLG on signal knee 38 0.79 0.41 19 0.95 0.23 163 2.43∗∗∗∗ 0.68 25 2.16∗∗∗∗ 0.55
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BMI: body mass index, KLG: Kellgren-Lawrence grades, VAS: visual analog scale, WOMAC: Western Ontario & McMaster Universities Osteoarthritis Index.

3.4. Associations between biomarkers and demographics

To investigate the relations between biomarkers and demographic variables, Spearman's correlation was performed with biomarkers (sCTX-II either measured in serum or urine, uCTX-II measured in urine) as the dependent variable, and gender, age, and BMI as independent variables. Both sCTX-II levels in urine and uCTX-II levels were significantly associated with gender (Table 2), which was consistent with the previous report [30,31]. In contrast, sCTX-II levels in serum had no association with gender but negatively associated with age. In the corresponding plots (Fig. 3), mean concentrations of the sCTX-II tended to decrease with age, with minimum values seen in participants aged 70–79. On the contrary, the uCTX-II levels slightly elevated with increasing age in both genders from 40 years and upwards. Particularly, the uCTX-II concentration in women aged 70–79 was significantly higher in comparison to that in men (P < 0.05, Fig. 3). In the corresponding plots (Fig. 3), mean concentrations of the sCTX-II tended to decrease with age, with minimum values seen in participants aged 70–79. On the contrary, the uCTX-II levels slightly elevated with increasing age in both genders from 40 years and upwards. Particularly, the uCTX-II concentration in women aged 70–79 was significantly higher in comparison to that in men (P < 0.05, Fig. 3).

Table 2.

Associations between uCTX-II, sCTX-II, and demographic variables as assessed by Spearman's correlation. Spearman's correlation coefficients (r) and significance values (p) are reported per demographic variable.

Age Gender BMI
uCTX-II (urine) r 0.076 −0.204 0.123
p value 0.2340 0.0013 0.0551
sCTX-II (serum) r −0.151 0.040 −0.038
p value 0.0185 0.5356 0.5584
sCTX-II (urine) r 0.171 −0.285 0.089
p value 0.0081 < 0.0001 0.1727
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BMI: body mass index, CTX-II: C-terminal telopeptide of type II collagen.

Fig. 3.

Fig. 3

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SCTX-II (A) and uCTX-II (B) levels per 10-year age category in men (open circle) and women (filled circle) aged 40–79. For sCTX-II, the levels decreased slightly in both genders during the examined age range. For uCTX-II, men and women showed comparable uCTX-II levels after the age interval of 50–59 years. The uCTX-II increase was less apparent in men and was generally lower in men than in women throughout the concerned age category. Results were presented as the mean and standard error of the means (SEMs) and were compared with Student's two-tailed t-test. ∗p < 0.05. uCTX-II: urinary C-terminal telopeptide of type II collagen.

3.5. Relations between sCTX-II and uCTX-II

To evaluate the specificity of the sCTX-II assay, the correlation between sCTX-II and uCTX-II was analyzed by Spearman's correlation. The sCTX-II levels in serum were not associated with uCTX-II in urine and sCTX-II in urine (r = 0.0119, r = −0.0225, respectively, Table 3). Interestingly, sCTX-II levels in urine were significantly correlated with uCTX-II (r = 0.4600, Table 3), suggesting that to some degree the sCTX-II ECLIA detected the same analytes in urine as uCTX-II did.

Table 3.

Associations between uCTX-II and sCTX-II as assessed by Spearman's correlation analysis. Spearman's correlation coefficients and p values are reported.

uCTX-II (urine) sCTX-II (serum) sCTX-II (urine)
uCTX-II (urine) r – 0.0119 0.4600
p value 0.6487 < 0.0001
sCTX-II (serum) r 0.0119 – −0.0225
p value 0.6487 0.9433
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3.6. Associations between sCTX-II/uCTX-II and clinical variables

uCTX-II concentrations were significantly elevated in the subjects with higher KLG compared to the ones with lower KLG (Fig. 4, B+D). By contrast, the sCTX-II levels decreased gradually when the KLG increases although not significantly (Fig. 4, A+C). uCTX-II was able to discriminate the ROA patients from the non-OA controls (Fig. 4, F). Meanwhile, there was a significant difference in uCTX-II between ROA+S group and healthy control (P < 0.05, Fig. 4, H). However, sCTX-II did not demonstrate such abilities (Fig. 4, E+G). In addition, we assessed the associations between pain scores and biomarkers. Unfortunately, neither sCTX-II nor uCTX-II was associated with VAS pain (Fig. 4, I+J).

Fig. 4.

Fig. 4

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Serum CTX-II levels (A+C+E+G+I) and urinary CTX-II levels (B+D+F+H+J) depending on the KL grades and VAS pain scores. Data are shown as mean ± SEM, and the non-parametric Kruskal-Wallis test is used to test for significance. The p-values are adjusted for age, BMI, and sex. Asterisks indicate the following: ∗P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. BMI: body mass index; CTX-II: cross-linked carboxyl-terminal telopeptide of type II collagen; KLG: Kellgren-Lawrence grades; ROA + S: radiographic OA with pain; ROA-S: radiographic OA without pain; VAS: visual analog scale.

We further examined the relationship between biomarker tertiles (sCTX-II and uCTX-II) and the difference in clinical parameters. The KL grades, VAS pain, WOMAC pain, stiffness and function scores were significantly higher in the highest uCTX-II tertile (T3) compared to the lowest tertile (T1) (Supplementary Fig. 1-B+D+F+H+J, P < 0.0001, P < 0.05, P < 0.01, P < 0.001, P < 0.001, respectively). In contrast, a reverse trend was observed in sCTX-II tertiles although no significances were observed (Supplementary Fig. 1-A+C+E+G+I).

4. Discussion

We here developed an immunoassay, which is capable to analyze CTX-II concentrations in human sera. The main findings were: 1) The specificity of the sCTX-II ECLIA was evaluated in a bovine cartilage explant model. 2) sCTX-II levels in serum were not associated with uCTX-II in urine although sCTX-II levels in urine were significantly correlated with uCTX-II. 3) uCTX-II correlated with age and gender while sCTX-II did not. The age-dependent pattern in uCTX-II showed noteworthy differences with that of sCTX-II. 4) sCTX-II cannot demonstrate any clinical relevance in a cross-sectional OA cohort as uCTX-II did.

We used the same monoclonal antibody from previous uCTX-II ELISA for sCTX-II ECLIA development. As previously reported, the monoclonal antibody demonstrated high specificity against EKGPDP. It had no cross-reactivity with the synthetic peptides EKGPD and REKGPD, which were lacking one C-terminal proline (P). Meanwhile, the antibody did not recognize the peptides EKGPDPL and KGPDPL that were extended with a C-terminal leucine (L) [6]. In the bovine explants study, OT group resulted in significantly higher concentrations of CTX-II in the late phase of culture compared to the vehicle group. This finding was in line with the literature [32,33]. Inhibition of MMPs by GM6001 attenuated the CTX-II levels compared to the OT group, as described by Sondergaard et al. [34]. The absence of CTX-II at the early and mid-phase of culture indicates lower MMP activity in comparison to those of later time points, which was also in agreement with previous observations [33,34]. In the C4Pain cohort, our data confirmed that uCTX-II are elevated in OA patients with severe KLG and the uCTX-II assay shows the ability to distinguish the ROA patients from the non-OA subjects, which agree with previous findings [17,[35], [36], [37]]. However, our results indicate that the serological forms of CTX-II do not have the same clinical relevance as the urinary forms do. Previously, we reported that the plasma CTX-II showed no clinical relevance in this OA cohort [38], suggesting the CTX-II profiles were consistent in serum and plasma. Unexpectedly, the sCTX-II levels were declining with increasing KLG although no significance. One possible explaination is that some OA patients with higher KLG lost most of the articular cartilage, resulting in less type II collagen (and CTX-II) present in knee and blood. In the other hand, the corresponding uCTX-II levels can still elevate due to the accumulation effect in urine. Comparisons of correlations between uCTX-II and sCTX-II with the correlations between the uCTX-II and sCTX-II (urine) proved that sCTX-II is not just uCTX-II. Furemore, we developed a competitive sCTX-II ECLIA as well in that the uCTX-II assay is in competitive format. Unfortunately, the sCTX-II competitive ECLIA showed almost the same profile as the sandwich format (see Supplementary Fig. 2). Taken toghether, we believe the reason the sCTX-II assay is not clinically applicable has nothing to do with the formats or sensitivities of the sCTX-II assays.

We suspect the discordance of serum and urine CTX-II profiles is due to the fact that the structure and sequences of serological CTX-II have not been thoroughly studied yet. Eyre et al. reported that the CTX-II fragments in blood comprised of EKGPDP, LGPREKGPDP, FAGLGPREKGPDP, and IDMSAFAGLGPREKGPDP, etc. In contrast, the urinary counterparts only consisted of EKGPDP [5]. The similar profiles measured by uCTX-II and sCTX-II (measured in urine) indicate that the form of CTX-II in urine is mainly dimeric, although uCTX-II has been speculated to be monomeric [39]. Recently, Henrik et al. developed a CTX-II+2 assay (EKGPDPLQ1237), which was speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. The EKGPDPLQ1237 assay was shown to be relevant in diseases with pathological osteoclast activity and cartilage degradation although no clinical relevance was observed in human blood, or urine from healthy subjects or arthritis patients [40]. Therefore, we believe that the reason blood CTX-II assay is not clinically relevant is that there are several distinct types of CTX–II–containing fragments in the blood (see Supplementary Fig. 3). Such fragments may originate from distinct pathological processes of cartilage degradation, which complicates the clinical application of serum CTX-II or alike assay. In contrast, only one type of CTX-II fragment (EKGPDP) was found in urine (see Supplementary Fig. 3), where all serological forms of CTX-II (EKGPDP, LGPREKGPDP, FAGLGPREKGPDP, and IDMSAFAGLGPREKGPDP) are further degraded to EKGPDP form and enriched when the fragments are transported through kidney [41].

However, some limitations in the present study should be noted. First, this study did not measure sCTX-II in synovial fluid, therefore it is unknown if sCTX-II concentrations in synovial fluid are correlated with the levels in the blood. Like urinary CTX-II, the blood CTX-II does not directly represent the local environment of the joint and instead represent input from the entire body. Second, the relatively small number of subjects in this study also implies that the findings need to be validated in larger populations. Lastly, this clinical cohort is cross-sectional and retrospective in nature, thereby longitudinal studies are required to further investigate the clinical relevance of the sCTX-II assay.

5. Conclusions

We put forward a serological CTX-II immunoassay for the first time and investigated its value to reflect systemic type II collagen degradation in OA patients. This discrepancy of sCTX-II and uCTX-II is probably caused by the structure, sequences, and abundance difference of CTX-II in blood and urine. Further efforts are required to confirm the difference between serological CTX-II and urinary CTX-II and target the right epitope of CTX-II in blood.

Authors' contributions

YYL and YH carried out the study procedures and drafted the manuscript. ACBJ, and MK designed the study. All authors approved the final manuscript.

Funding

The work was financed by the Danish Research Foundation.

Declaration of competing interest

The authors have no competing interests. YYL, YH, ACBJ, and MK are full-time employees of Nordic Bioscience.

Acknowledgments

We would like to thank IDS (UK) for providing the antibody. Furthermore, we thank Sabine Hoielt for collecting the OA patients' samples.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ocarto.2020.100082.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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