Mouse Proteinase 3 (PR3) ELISA
Proteinase 3 (PR3), also known as myeloblastin, Wegener autoantigen, PRTN3 and NP- 4, is one of the hematopoietic serine proteases localized in the primary granules of polymorphonuclear neutrophils (PMNs). The primary function of PR3 is recognized as to participate in direct intracellular killing of phagocytosed pathogens in phagolysosomes and degradation of extracellular matrix components at inflammatory. PR3 has also been proven to be able to process some pro-inflammatory cytokines, such as IL- 1β, IL-18 and TNF-α, activate mitogen activated protein kinase (MAPK) signaling through proteinase activated receptor-1 (PAR1), and induce endothelial cell apoptosis through NF-κB signaling pathways. PR3 is identified as the target autoantigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in Wegener granulomatosis. Increased PR3 levels have been reported in patients with acute myocardial infarction, and in subjects with type 1 diabetes.
PRINCIPLE OF THE ASSAY
This assay is a quantitative sandwich ELISA. The microtiter strips are precoated with a polyclonal antibody specific for mouse PR3. Standards and samples are pipetted into the wells and any mouse PR3 present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidise (HRP) labelled polyclonal antibody specific for mouse PR3 is added to the wells. After wash step to remove any unbound reagents, an HRP substrate solution is added and colour develops in proportion to the amount of mouse PR3 bound initially. The assay is stopped and the optical density of the wells is determined using a microplate reader. Since the increase in absorbance is directly proportional to the amount of captured mouse PR3, the unknown sample concentration can be interpolated from a reference curve included in each assay.
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.
Absorbance (450 nm)