Pre-treatment of synovial fluid enable precise and accurate measurement of neo-epitope biomarkers

Purpose: The synovial fluid (SF) provides lubrication and essential nutrients for cartilage, as well as removing waste products from metabolizing cells in the joint. Biomarkers measured in SF may provide a direct measure of cartilage and synovial turnover of the joint from where the SF was retrieved. In contrast, serum measurements of joint-related biomarker may reflect cartilage and synovial turnover of multiple joints. However, the matrix of the synovial fluid is different than the matrix of serum or plasma as it contains a high degree of hyaluronic acid that makes the SF viscous and sticky. The matrix effect of synovial fluid is therefore also different, which makes it challenging to measure small neo-epitope biomarkers herein. Accuracy and precision are key in measuring biomarkers in all types of samples. Thus, it is critical to thoroughly test and validate a biomarker in the biological matrix that it is being measured in. Accordingly, the purpose of this study was to optimize and validate the measurement of four joint-related neo-epitope biomarkers, CRPM (chronic tissue inflammation), C2M and AGNx1 (cartilage degradation), and C3M (connective tissue remodeling) in SF. Methods: Human and bovine SF was centrifuged and pre-treated with 0.4 mU chondroitinase ABC and 0.4 mU Endo-β-galactosidase pr. μL synovial fluid for 3 hours at 37°C prior to measurement of the biomarkers. Each biomarker tested for dilution recovery and spike-in recovery. Dilution recovery: Samples were diluted in a 2-fold dilution in the assay buffers from the biomarker measured. C3M enrichment of synovial fluid: The standard peptide from C3M assay were added to the untreated SF samples. The SF samples were then treated and used for dilution recovery as described above. Spike-in recovery: The standard peptide from the biomarker measured were diluted in a 2-fold dilution and four points were mixed 1+1 with bovine SF or assay buffer. Acceptance range for dilution- and spike-in recovery was 80–120%. As control SF samples were mixed 1+1 in assay buffer and also included. The biomarkers were measured with competitive ELISA specific for C2M, C3M, CRPM, and exAGNx1 (NITEGE). Results: The deglycosylated pre-treated SF samples had a dilution recovery between 98–120% compared to neat for C2M (Fig.1A), 89–110% for CRPM (Fig.1C), and 85–93% for AGNx1. The levels of C3M were just around lower limit of measuring range (LLMR) and the dilution recovery could not be determined in the SF. We therefore enriched the synovial fluid with C3M to measure dilution recovery in the SF matrix. When enriched for C3M the dilution recovery was 99–104%. Additional the spike-in recovery was measured to test the precision of the assay in synovial fluid. The spike-in recovery for C2M was 97–106% (Fig.1B), for CRPM within measuring range 114-107% (Fig.1D), for AGNx1 97–121%, and 109-122% for C3M within measuring range. Conclusions: Pre-treatment with 0.4 mU chondroitinase ABC and 0.4 mU Endo-β-galactosidase pr. μL SF enabled accurate and precise measurement for the four joint-related biomarkers: C2M, C3M, CRPM, and AGNx1. These data show that it is possible to reliably measure neo-epitope biomarkers of cartilage and synovial turnover in the synovial fluid. The DBOARD consortium supported this study.