Dog NEUTROPHIL ELASTASE SPARCL™ Assay | NEL-SP-4

INTRODUCTION

Neutrophil elastase (NE) is a serine protease that is secreted by neutrophils. In humans, increased serum levels have been associated with colorectal cancer (ref 1). Increased sputum levels have been found in COPD patients with bacterial infection (ref 2) and in patients with bronchiectasis (ref 3)

PRINCIPLE OF THE ASSAY

The dog NE SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 4) assay uses NE antibodies that were developed at Life Diagnostics, Inc. One is conjugated to horseradish peroxidase (HRP), the other to acridan, a chemiluminescent substrate. When the HRP and acridan conjugates bind to NE they are brought into proximity. With the addition of hydrogen peroxide, HRP catalyzes oxidation of proximal acridan molecules causing a flash of chemiluminescence. Acridan conjugated antibodies distant from HRP (not bound to NE) produce no signal. This principle allows the development of a homogeneous assay that allows rapid measurement of NE. Standards and diluted samples are mixed with HRP and acridanconjugates in the wells of the 96-well SPARCL™ plate2 provided with the kit. After incubation for 30 minutes on a shaker at 25°C and 150 rpm, the plate is placed into a luminometer. Trigger solution containing hydrogen peroxide is injected into each well and luminescence is immediately measured. The concentration of NE is proportional to luminescence and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Anti-NE HRP conjugate Store ≤ -70°C

• Anti-NE acridan conjugate Store ≤ -70°C

• NE stock (lyophilized), 2 vials Store ≤ -70°C

• Diluent; CSD50-1, 2 x 50 ml

• Trigger solution; TS7-1, 7 ml

• White SPARCL™ plate (12 x 8-well)

• Clear untreated 96-well plate

Materials required but not provided:

• Precision pipettes and tips

• Polypropylene microcentrifuge tubes

• Vortex mixer

• Plate incubator/shaker

• Luminometer capable of simultaneous injection/measurement

• Curve fitting software

STORAGE

Store the HRP conjugate, acridan conjugate and haptoglobin stock at -70°C (they may be stored at -20°Cfor one week). The remainder of the kit should be stored at 2-8°C. The SPARCL™ plate should be kept in a sealed bag with desiccant and antioxidant. The kit will remain stable for at least six months from the date of purchase, provided that the components are stored as described above

GENERAL INSTRUCTIONS

1. Please take the time to completely read all instructions before starting your assay. Contact us if you need clarification.

2. All reagents used in the assay should be allowed to reach room temperature (25°C) before use.

3. It is important that standards and samples be added to the SPARCL™ plate quickly. If testing large numbers of samples, rather than pipetting standards and samples directly into the white SPARCL™ plate using a single channel pipettor, we recommend the following. First, pipette an excess volume of standards and samples into appropriate wells of the clear 96- well plate. Then use an 8- or 12-channel multipipettor to quickly and efficiently transfer 50 ml aliquots to the appropriate wells of the white SPARCL™ plate. The wells of the clear plate hold a maximum volume of 300 ml.

4. Follow the sequence of events below when running the assay.