CORTISOL SPARCL™ Assay | CORT-SP

FOR RESEARCH USE ONLY

NOT FOR USE IN HUMAN OR VETERINARY DIAGNOSTICS

INTRODUCTION

Cortisol is a glucocorticoid steroid hormone. It is produced in the adrenal cortex and released into the blood in response to stress. It aids in regulation of glucose levels, immune response and cardiovascular function.

PRINCIPLE OF THE ASSAY

The cortisol SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 1) assay uses a competitive format. Cortisol present in samples competes for binding of cortisol-HRP to a cortisol antibody that is conjugated to acridan, a chemiluminescent substrate. When cortisol-HRP binds to anti-cortisol-acridan, HRP and acridan are brought into close proximity. With the addition of hydrogen peroxide, HRP catalyzes oxidation of proximal acridan molecules causing a flash of chemiluminescence. Acridan conjugated antibody that is not bound to cortisol-HRP produces no signal. This principle allows the development of a no-wash assay that allows rapid measurement of cortisol concentrations. During the assay, HRP-cortisol is first added to wells of a 96-well SPARCL™ plate2. Cortisol standards and diluted samples are then added. After brief mixing, anti-cortisol-acridan is added and the plate incubated on a shaker at 25°C and 150 rpm. After thirty minutes 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 cortisol is inversely proportional to luminescence and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Cortisol-HRP stock Store ≤ -70°C

• Anti-cortisol acridan stock Store ≤ -70°C

• Cortisol stock3 Store ≤ -70°C

• 10 ml Dissociation buffer; SDB10-1, 10 ml

• 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 tubes

• Vortex mixer

• Plate incubator/shaker

• Luminometer capable of simultaneous injection & measurement

• Curve fitting software

STORAGE

Store the HRP conjugate, acridan conjugate and CRP 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.