1. P021
High sensitivity Pro-C2, a novel sensitive, blood-based biomarker for measuring cartilage formation
Yun Y. Luo1, Yi. He1, Natasja S. Gudmann1, Anne S. Siebuhr1, Sabine. Hoielt1, Morten A. Karsdal1, Anne C. Bay-Jensen1
1 Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730, Herlev, Denmark
arthritic disease
METHODS
BACKGROUND
Type II collagen is the major collagen found in cartilage and is expressed in two forms: IIA and IIB. Type IIA procollagen propeptide (PIIANP) differs from Type IIB procollagen propeptide (PIIBNP) with the presence of exon 2 encoding a cysteine-rich globular domain. PIIBNP is mainly liberated during type II collagen formation in healthy adults whereas PIIANP is synthesized by chondroprogenitor cells in in embryonic and diseased cartilage. PIIBNP may serve as a specific arthritis biomarker that reflect the rate of type II collagen synthesis. (Fig. 1)
The N-terminal specific anti-PIIBNP monoclonal antibody (NB443-2E5) was characterized by ELISA and western blot. A PIIBNP competition electrochemiluminescence immunoassay (ECLIA), hsPro-C2, was developed on the MesoScale Discovery (MSD) platform (Fig. 2). The technical performance of the assay was evaluated according to standard operating procedures (SOPs). The serum levels of hsPro-C2 and commercial PIIANP (Merck-Millipore) as course of age in healthy Black male individuals was studied. The correlation of both assays was investigated in sera from Osteoarthritis, Rheumatoid arthritis, Pediatric, and healthy adult donors. Mean values and standard error of the mean (SEM) were compared using one-way ANOVA assuming normal distribution. Significance levels are indicated by asterisks; *P < 0.05, **P < 0.01.
AIM
To improve the sensitivity of existing PIIBNP immunoassay (Pro-C2) by using electrochemiluminescence technology for usage in human serum as a specific marker of cartilage formation.
hsPro-C2 ECLIA shows superior technical performance over ProC2
Structures of PIIANP & PIIBNP
Pro-C2 antibody is truly specific for PIIBNP
Figure 1. Schematic illustration of PIIANP and PIIBNP (Pro-C2). Type II procollagen is synthesized in two splice forms, type IIA and IIB, as the result of alternative splicing of exon 2 in N-propeptide region. The polyclonal antibody employed in the PIIANP ELISA recognizes the region encoded by exon 2. The monoclonal antibody utilized in Pro-C2 only recognizes the N-terminus of PIIBNP after the removal of the signal peptide.
Assay name
Detection Range
(ng/mL)
Lower Detection of Limit (ng/mL)
Intra- CV(%)
Inter- CV(%)
Pro-C2
Competition ELISA
2.5-40
1.0
9.2
10.8
hsPro-C2 Competition ECLIA
0.13
5.4
5.5
PIIANP Competition ELISA 30
4.5
6.7
Figure 3. Characterization of anti-PIIBNP antibody. (A) Peptide specificity test. The signal of NB443-2E5 was displaced by increasing concentration of PIIBNP selection peptide (QDVRQPGPKG), but not by the elongated (GQDVRQPGPKG) or cross-reaction peptide (QDVQPGPKG). (B) (C) Western blot analysis. Pooled serum from patients with OP (lane 2, 7, 11, 16), pediatric (lane 3, 12), healthy adult (lane 4, 13), patients with OA (lane 5, 14), patients with RA (lane 6, 15) and amniotic fluid (lane 8, 17) were incubated with anti-PIIBNP monoclonal antibody NB443-2E5 (lane 2-8) or normal mouse serum (lane 7, 16). PIIBNP was present as three forms with molecular weights of about 80, 24, and 16 KDa respectively, in human amniotic fluid and one form with MW of 80 KDa in serum. The anti-IIB antibody pre-incubated with selection peptide (C) did not anymore recognize the bands suggesting that the bands are indeed related to PIIBNP. The asterisks point to non-specific bands.
Table 1. The technical performance of assays used in this study. The lower limit of detection was computed as 3 Standard Deviation (SD) of the mean of 21 zero standards. Intra-CV% (within plate) and Inter-CV% (between plates) were calculated as the mean variation of 5 samples in 10 independent runs.
PIIBNP is not associated with PIIANP
hsPro-C2 ECLIA is consistent with Pro-C2 ELISA
How does hsPro-C2 ECLIA work?
Figure 6. Comparison between hsPro-C2 and PIIANP serum levels in randomly selected OA (A), RA (B), pediatric (C), and healthy adults samples (D); ns=non-significant difference. Pearson’s correlation coefficient (r), a regression line (solid) are indicated.
Figure 4. Comparison between hsPro-C2 ECLIA and Pro-C2 ELISA levels in nine randomly selected bovine explants supernatant and eight human serum. Pearson’s correlation coefficient (r), a regression line (solid) are indicated.
Figure 2. Basic principle of hsPro-C2 competition electrochemiluminescence immunoassay (ECLIA). 1) High binding carbon electrodes in the bottom of Streptavidin microplates allow for easy attachment of biotinylated PIIBNP antigen (10X greater binding capacity than polystyrene). 2) Electrochemiluminescent labels (SULFO-TAG) that are conjugated to detection antibodies allow for ultra-sensitive detection. 3) Electricity is applied to the plate electrodes by an MSD instrument leading to light emission by SULFO-TAG labels. 4) Light intensity is then measured to quantify analytes in the sample. (Adapted from MSD LLC.)
Non-significant difference as course of
age in both hsPro-C2 & PIIANP assays
Figure 5. Box-Whisker plots of the distribution of serum hsPro-C2 levels (A) and PIIANP (B) in healthy Black male adults. From the bottom up, the box indicated the 25th, 50th (median) and 75th percentiles. The tips of the whiskers showed the reference range limits (10th and 90th percentiles). The results presented above demonstrated that both Pro-C2 and PIIANP concentrations remained stable among any population of men (P = ns, Turkey analysis).
CONCLUSION
We have described a second-generation PIIBNP quantification assay when converting from a traditional ELISA format to the ECLIA format on the MSD platform. We have here shown how the use of new platform has resulted in a seven-fold increased sensitivity, allowing for quantification in serum. In our previous study, the antibody selected for the assay had no cross-reactivity with PIIANP nor mouse/rat PIIBNP. To our knowledge, we developed for the first time an serological immunoassay for human PIIBNP, which has no association with PIIANP. Further clinical validation of this novel assay in arthritis diseases is warranted.