Proteinase 3 (PR3) Summary
Active PR3 levels in sputum have been shown to correlate with clinical markers of inflammation in respiratory diseases including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Measurement of active PR3 in sputum may aid in the understanding several pulmonary diseases. The development of a novel activity-based immunoassay may help to determine the relative potential contribution of PR3 to lung damage and the implications in human airway disease.
The molecular mechanisms that drive chronic lung disease are heterogenetic. To evolve past a “one drug fits all approach”, and towards a more individualized and precise approach, pharmaceutical companies are focusing on drugs that address each etiology within a disease. Hence, endotypes, which are the distinct pathobiological mechanisms driving disease, have come to the forefront in drug design. The endotype approach is also needed for assessing individuals for drug development.
While many cell types can be accounted for in a diseased lung, there is usually one cell type that is the predominate driver of disease. This cell type can vary within a disease and can also change over the course of the disease. This makes endotyping an essential step to identify the distinct inflammatory cell type driving the disease in order to study the disease.
COPD, bronchiectasis, and asthma are most commonly characterized as eosinophilic or neutrophilic endotypes. Neutrophil elastase and proteinase 3 are proteases produced by neutrophils. When released, they facilitate a cycle of tissue damage and inflammation, driving pathology and disease. Measuring the activity levels of neutrophil elastase provides a specific and accurate assessment of neutrophil-driven disease to determine individual endotypes. This makes neutrophil elastase activity measurement an effective tool for drug development studies involving targets that inhibit neutrophil activity.
In addition to endotyping, neutrophil elastase measurement can function as a clinical trial endpoint for general inflammation in lung disease. Inflammation is a hallmark of chronic lung diseases and an important piece to understand when evaluating drug effectiveness. Decreasing lung-specific inflammation is a key strategy for pharmaceutical targets. Currently, the FEV1/FVC ratio is the primary endpoint during pulmonary drug development studies, but does not capture a complete picture of a disease state. Cytokine measurements are frequently recorded, but may not be specific to lung inflammation. Mechanistic markers of inflammation, like neutrophil elastase, can be detected earlier and indicate improvements in outcomes.
ProAxsis offers products that have proven to be valuable in enrolling, stratifying, and studying effectiveness of respiratory drugs in development. The ready-to-use kits offer a sensitive, reliable, and accurate read-out of neutrophil elastase and proteinase 3 activity in sputum or bronchoalveolar lavage (BAL). Neutrophil elastase and proteinase 3 activity levels correlate with disease severity, time to next exacerbation, and bacterial load. From an endotyping perspective, they are also global mechanistic markers of neutrophil-driven inflammatory states.
For more about PR3 as a neutrophil serine proteases (NSP) and biomarker, read more here >>>>
Principle of the Assay:
The ProteaseTag® Active PR3 Immunoassay utilizes ProteaseTag® technology to specifically detect and quantify active PR3. During a short incubation, the PR3-Tag is coated onto the immunoassay plate before washing to remove excess. Standards and samples are added, and the PR3-Tag will interact with the active site of PR3 during a second incubation step. Active PR3 present in the solutions will irreversibly bind to the PR3-Tag while latent and inhibitor bound PR3 will be removed by washing. A horseradish peroxidase (HRP) conjugated anti-PR3 antibody is added to each test well and incubated. This detection antibody attaches to bound PR3 with unbound antibody subsequently removed by washing. A color forming substrate containing tetramethylbenzidine (TMB) is added to each test well and reacts with HRP to generate a blue colored product. This enzymatic reaction is subsequently stopped by the addition of acidic stop solution to each test well (a yellow solution is formed). The color intensity (absorbance) is read at 450 nm using a plate reader.
Freezer Box Components (stored at -20°C)
|PR3-Tag||1 vial containing 30 uL (concentration 1mM)
of capture probe in DMF
|PR3 Standard||2 vials containing 10 uL of active PR3
(concentration 0.05 mg/mL)
*Each vial can only be used once
|Secondary PR3 Antibody||1 vial containing 30 uL of anti-mouse IgG
conjugated to HRP in a stability solution
Main Box Components (stored at 2-8°C)
|Immunoassay Plate||96 well strep coated microtiter plate (pre-blocked)|
|PR3 Wash Buffer A Concentrate||32 mL of 25-fold concentration of buffered surfactant with preservatives|
|PR3 Wash Buffer B Concentrate||24 mL of 10-fold concentration of buffered surfactant with preservatives|
|Primary PR3 Antibody||1 vial containing 30 uL anti-PR3 mouse monoclonal antibody in a stability solution|
|TMB Substrate||12 mL of tetramethylbenzidine solution|
|Stop Solution||6 mL of 2N sulfuric acid|
|Plate Sealers||5 adhesive strips|
Format: 96 well
Sample: Sputum, bronchial alveolar lavage fluid (BALF)
Range: 7.81 –500 ng/ml
LOD: 2.35 ng/ml
Run Time: 230 min of incubation