Tartrate-resistant acid phosphatase (TRACP) is a biomarker with multiple clinical and basic applications. It has long been used as a cytochemical marker to identify hairy cell leukemia cells and as a marker for differentiated cells of monocyte lineage including macrophages (MΦ), osteoclasts (OC), and dendritic cells (DC). The clinically relevant MΦ/OC/DC TRACP is called Type-5 acid phosphatase encoded by the Acp5 gene. TRACP consists of two isoforms (Type 5a and 5b) that have different biochemical properties, clinical significance and perhaps biological functions. TRACP5a circulates as a 35 kDa glycoprotein with an intact central regulatory loop peptide, which interacts with the active site. TRACP5b circulates as a disulfide linked heterodimer in which the loop peptide has been proteolytically processed to yield 16 kDa and 23 kDa fragments. Osteoclasts secrete TRACP5b during bone resorption making it a sensitive and specific serum biomarker for OC number and bone resorption rate. TRACP5a, but not 5b, is secreted by activated macrophages and dendritic cells. Elevated serum TRACP5a is emerging as a potential marker for systemic macrophages and chronic inflammation. The principal clinical application of serum TRACP immunoassay is to estimate isoform 5b activity as a marker of osteoclast number and bone resorption rate. This is done in commercial immunoassays by increasing the pH to a level selectively permissive for isoform 5b activity and / or by using a substrate selective for isoform 5b. The antibody used in this ELISA reacts with a critical epitope and binding to TRACP protein causes inactivation of the enzymatic activity.
The human TRACP5a ELISA kit is to be used for the in vitro quantitative determination of human TRACP5a in plasma and serum samples.
The human TRACP5a ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are incubated in microtiter wells coated with antibodies recognizing human TRACP5a.
Biotinylated tracer antibody will bind to captured human TRACP5a.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human TRACP5a standards (log).
The human TRACP5a concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.