Research Papers using IBEX ELISA assays

Liisa Kuhi ^1.2 Ann E Tamm ¹ Jaanika Kumm ^1,3 Kristel Järv ^1.3
Aare Märtson ^1.4 Agu O. Tamm ¹ Kalle Kisand ¹

1. Institute of Clinical Medicine, Faculty of Medicine, University of Tartu, 50406 Tartu, Estonia
2. Central Laboratory, Diagnostic Clinic, East-Tallinn Central Hospital, 10138 Tallinn, Estonia
3. Radiology Clinic, Tartu University Hospital, 50406 Tartu, Estonia
4. Traumatology and Orthopaedics Clinic, Tartu University Hospital, 50406 Tartu, Estonia

The objective of this study was to investigate the dynamics of the urinary collagen type II C-terminal cleavage neoepitope (uC2C) before and after total knee replacement (TKR) in rapid knee OA progressors. C2C in the urine was measured by IBEX-uC2C assay in 86 patients (mean age: 59.9 years) with symptomatic knee OA (kOA) undergoing TKR, assessed before surgery and 3 and 12 months after. The patients’ condition was determined by self-assessment questionnaires, by lower limb performance tests, and by radiography. In the preoperative period, the uC2C level was significantly higher in females than in males, and was associated with the radiographic severity of kOA. A weak correlation between the C2C and knee pain was observed in the whole group and in males, but not in females. The individual dynamics of uC2C after TKR were heterogenic. In general, uC2C increased three months after TKR, but fell to the preoperative level after 12 months. A higher preoperative uC2C implied the tendency to diminish as a result of TKR, and vice versa. TKR did not stop the degradation of Coll2 in the tissues in the majority of cases. The pre-TKR uC2C predicts the postoperative uC2C level. The uC2C dynamic seems to be sex-specific, so it could be considered a prospective pre- and post-TKR biomarker for progressive kOA.

1.1 PRINCIPLE OF PROCEDURE The C2C assay measures a neoepitope created by the cleavage of type II collagen by collagenases. This neoepitope that is created at the carboxy terminus of the three-quarter length piece of the primary cleavage site is recognized by the C2C monoclonal antibody which is specific for type II collagen. This assay is for in vitro research use only and has been optimized for analyzing human serum. It has also been used to analyze various types of samples and species. This assay has been designed to be used for comparative analysis only, and is not to be used for absolute diagnostic purposes. All samples must be treated the same way and in the recommended manner.

1.2 PRINCIPLE OF THE ASSAY This assay is a competitive immunoassay in a 96-well plate format using a mouse monoclonal antibody and a synthetic peptide to represent the neoepitope (Poole et al, 2004). This synthetic peptide is conjugated to a protein and pre-coated onto the C2C ELISA plate. C2C peptide standards and unknown serum samples are added to a polypropylene mixing plate, followed by specific mouse IgG antibody (C2C antibody formerly called the col 2 3/4 long antibody). This mixture is pre-incubated to allow antibody binding to the free C2C peptide. The pre-incubated samples are then transferred from the polypropylene mixing plate onto the C2C ELISA plate and incubated to allow the antibody to bind either to the immobilized peptide on the plate, to the C2C standards or to the endogenous neoepitope in serum samples. After washing the C2C ELISA plate, conjugated goat anti-mouse horseradish peroxidase (GAMHRP) is added, which binds to any mouse antibody on the C2C ELISA plate. After washing the C2C ELISA plate again, Tetramethylbenzidine substrate (TMB) is added to each well which react with HRP to form a blue product. The reaction is stopped and the signal amplified with an acid, which converts the product from a blue to a yellow colour that can be quantified at 450 nm. The optical density (OD) at 450 nm is inversely proportional to the amount of neoepitope present in the sample.

Liisa Kuhi ^1.2 Ann E Tamm ¹ Agu O. Tamm ¹ Kalle Kisand ¹

1. Department of Internal Medicine, Institute of Clinical Medicine, Faculty of Medicine, University of Tartu, 50406 Tartu, Estonia
2. Central Laboratory, Diagnostic Clinic, East-Tallinn Central Hospital, 10138 Tallinn, Estonia
3. Sports Medicine and Rehabilitation Clinic, Institute of Clinical Medicine, University of Tartu, 50090 Tartu, Estonia.

One of the unmet needs to be addressed is prognostic biomarkers for early knee osteoarthritis (kOA). We aimed to study the association of urinary collagen type-II C-terminal cleavage neoepitope (uC2C) with the emergence and progression of kOA. The longitudinal data of 330 subjects (aged 32-60 years) from an Estonian population-based cohort were used. The radiographic progression was evaluated by the grading system of Nagaosa et al. of knee compartments at baseline and three years later. The emerging kOA consisted of subjects with developing osteophytes or joint space narrowing, whereas kOA progressors showed aggravation of radiographic grade. Baseline uC2C levels were measured by the IBEX-uC2C assay. At baseline, the subjects were middle-aged (mean age, 47.6 years) and overweight (mean BMI, 28.0 kg/m²), and the majority of them (51.2%) had a diagnosis of kOA grade 1. Multiple logistic regression models adjusted for sex, age, and BMI were used for risk calculations. We demonstrate that increased uC2C accurately predicted the risk of emerging of kOA (OR = 5.87 (1.71-20.22); AUC = 0.79) compared with controls without radiographic kOA over 12 years. However, the most accurate prediction of progression by the biomarker was found in women (OR = 23.0 (2.2-245), AUC = 0.91). In conclusion, uC2C may be a promising candidate as a prognostic biomarker for kOA progression, particularly of emerging kOA in women.