Pr-3 Summary

Active Pr-3 levels in sputum have been shown to correlate with clinical markers of inflammation in respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease. Measurement of active Pr-3 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 Pr-3 to lung damage and the implications in human airway disease.

For more about Pr-3 as a neutrophil serine proteases (NSP) and biomarker, read more here >>>>

 

Principle of the Assay:

The ProteaseTag® Active Pr-3 Immunoassay utilizes ProteaseTag® technology to specifically detect and quantify active Pr-3. 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 Pr-3 during a second incubation step. Active Pr-3 present in the solutions will irreversibly bind to the Pr3-Tag while latent and inhibitor bound Pr-3 will be removed by washing. A horseradish peroxidase (HRP) conjugated anti-Pr-3 antibody is added to each test well and incubated. This detection antibody attaches to bound Pr-3 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

Pr-3 Standard 2 vials containing 10 uL of active Pr-3

(concentration 0.05 mg/mL)

*Each vial can only be used once

Secondary Pr-3 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)
Pr-3 Wash Buffer A Concentrate 32 mL of 25-fold concentration of buffered surfactant with preservatives
Pr-3 Wash Buffer B Concentrate 24 mL of 10-fold concentration of buffered surfactant with preservatives
Primary Pr-3 Antibody 1 vial containing 30 uL anti-Pr-3 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

Species:           Human

Sample:           Sputum, bronchial alveolar lavage fluid (BALF)

Range:             7.81 –500 ng/ml

LOD:                2.35 ng/ml

Run Time:        230 min of incubation