TESTOSTERONE SPARCL™ Assay | TEST-SP

FOR RESEARCH USE ONLY

NOT FOR USE IN HUMAN OR VETERINARY DIAGNOSTICS

INTRODUCTION

Testosterone is the male steroid sex hormone. It is produced primarily in the testes. It controls the development of male reproductive tissues and secondary sexual characteristics. In females the ovaries and adrenal glands are the major sites of production. Serum levels vary with age and are typically 10-20 times greater in males compared to females.

PRINCIPLE OF THE ASSAY

The testosterone SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 1) assay uses a competitive format. Testosterone present in samples competes for binding of testosterone-HRP to a testosterone antibody that is conjugated to acridan, a chemiluminescent substrate. When testosterone-HRP binds to anti-testosterone-acridan, HRP and acridan are brought into 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 testosterone-HRP produces no signal. This principle allows the development of a no-wash assay that allows rapid measurement of testosterone concentrations. During the assay, HRP-testosterone is first added to wells of a 96- well SPARCL™ plate2. Testosterone standards and diluted samples are then added. After brief mixing, anti-testosterone-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 testosterone is inversely proportional to luminescence and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Testosterone-HRP conjugate. Store ≤ -70°C

• Anti-testosterone acridan conjugate. Store ≤ -70°C

• Testosterone 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.