Background There is a medical need for biomarkers that reflect bone and cartilage turnover within the field of rheumatoid arthritis. This includes biomarkers which quantify cartilage turnover.
The N-terminal part of type II procollagen is expressed in two splice variants whereas the beta version (P2BNP) is the only one expressed in healthy cartilage of adults. We therefore hypothesized that P2BNP is of relevance during regeneration/repair of mature cartilage.
Objectives Our aim was to develop an ELISA (ProC2), which is specific for (P2BNP). Furthermore we wanted to investigate whether the assay could detect changes in collagen formation in an ex vivo bovine full-depth cartilage explants model (BEX).
Methods A competitive ELISA measuring P2BNP was developed applying a monoclonal antibody raised in mouse recognizing the N6-terminus of the N-terminal propeptide (sequence QDVRQPG) of collagen type II. The assay was technically valid. The range of the assay was 1.25-37nmol/L (intra-assay CV 6.87%, inter-assay CV 8.92%). Tests of linearity and analytic sensitivity, specificity, and sample stability were evaluated. Samples and antibody were incubated on streptavidin plates coated with biotin-QDVRQPG. EnVision was then added. Tetramethyl benzinidine (TMB) was applied and the TMB reaction was stopped by H2SO4. The absorbance was measured at 450 nm with 650 nm reference.
BEX explants (approx. 12mg) isolated from a bovine femur condyle was cultured for 3 weeks in serum free DMEM/F12 medium, with 3 weekly changes and in presence of following: transforming growth factor (TGF)-β, fibroblastic growth factor (FGF)-2, and insulin like growth factor (IGF)-1. Each growth factor in concentrations of 100, 10, 1, 0.1, 0.001ng/ml. Supernatant was collected. Data are shown as mean [95%>CI] or (SD). Two tailed Mann Whitney test compare treatment with vehicle. Viability of BEX was tested by Alamar blue at termination day.
Results The ProC2 assay was sensitive and specific for human and bovine P2BNP seen by 100% inhibition of signal with 150nmol/L selection peptide. The assay did not cross react with the corresponding rat/mouse or human alpha splice variant. P2BNP was quantified in human amniotic fluid (163-187,7nmol/L) and FBS (851-901nmol/L) and showed excellent linearity (dilution range 1:2-1:64 and 1:20-1:160 with 100% (SD 7.6) recovery). In contrast, serum P2BNP levels in healthy adults were below lower detection limit. No significant matrix effect was observed in any samples. TGF-β and IGF-1 significantly induced P2BNP secretion in BEX in a dose-dependent manner compared to the untreated (see figure). This association was not found in BEX stimulated by FGF-2.
Conclusions To our knowledge this is the first assay which is able to evaluate P2BNP excretion. The ProC2 assay is a promising and novel marker of collagen type II formation. The assay was both sensitive and specific for the beta-splice variant of collagen type II. In addition the assay was able to differentiate between P2BNP level in supernatant of BEX explants stimulated by either TGF-β or IGF-1 compared to untreated.
Disclosure of Interest None declared