This assay employs a two-site sandwich ELISA to quantitate CTX-I in samples. An antibody specific for CTX-I has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any CTX-I present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for CTX-I is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of CTX-I bound in the initial step. The color development is stopped and the intensity of the color is measured.

The carboxyterminal telopeptide of type I collagen (ICTP) is an indicator of degradation of type I collagen. ICTP antigen consists of a trivalent collagen cross-link joining three polypeptide chains of which two are α1 chains of one collagen molecule while the third is derived from either an α1 or an α2 chain of the helical region of another molecule (Fig. 1) (Risteli et al. 1993). The major determinant for antigeneity must contain two phenylalanine-rich regions, which is only possible if the cross-link is trivalent in nature (Sassi et al. 2000). In osteoclasts, catepsin K cleaves the trivalently cross-linked ICTP structure at two sites between the phenylalanine-rich region and the cross-link, thereby destroying any reactivity with ICTP antibodies (Sassi et al. 2000). Therefore degradation of type I collagen in bone is not measurable by the present ICTP-test. ICTP is cleared from circulation by the kidneys and has a molecular mass of about 12 000-20 000 (Risteli et al. 1993).

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.

Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.