Bone Abstracts

Tartrate resistant acid phosphatase (TRAP) consists of two isoforms TRAP 5a and TRAP 5b suggested to exert different functions and clinical relevance. TRAP 5a is a 35 kDa monomer and a potential marker of inflammatory conditions e.g. atherosclerosis and rheumatoid arthritis. TRAP 5b, a heterodimer of 16 and 23 kDa generated by proteolytic cleavage of a repressive loop in TRAP 5a, is used as marker for osteoclast numbers/bone resorption and osteoclast-related pathological conditions. The different functions/pathophysiological relevance of the two isoforms raise the need for a method that separately quantifies protein levels of each isoform. Here we report a sandwich ELISA assay quantifying protein concentrations of TRAP 5a and TRAP 5b in one and the same sample.

The repressive loop present on TRAP 5a but not TRAP 5b was used as immunogen to generate a monoclonal antibody (mAb) 46 specific for TRAP 5a. Full length TRAP 5a was used to raise mAbs recognizing both isoforms. mAb 46 was used as capture antibody in a sandwich ELISA for quantification of TRAP 5a from a TRAP 5a/TRAP 5b protein mixture. After capturing of TRAP 5a, the supernatant free of TRAP 5a was used in another ELISA composed of a capturing mAb recognising both isoforms to quantify TRAP 5b protein. The assay was tested with TRAP 5a/TRAP 5b mixtures and TRAP 5a protein was successfully separated from TRAP 5b protein. In healthy human serum samples the mean concentration of TRAP 5a protein and TRAP 5b protein were determined to be 3.6±0.76 and 0.65±0.31 ng/ml, respectively.

In summary, this novel TRAP 5a/5b sandwich ELISA specifically measures TRAP 5a and 5b protein separately in one and the same sample and thus increases the usefulness of TRAP 5a and 5b protein as markers for different pathological conditions.