Rat Myoglobin SPARCL™ Assay | MYO-SP-2

INTRODUCTION

Myoglobin is a 17 kDa heme protein that is expressed in both cardiac and skeletal muscle. After cardiac muscle injury myoglobin is one of the first biomarkers to increase above baseline values. In the absence of skeletal muscle injury, it can be used as a biomarker to assess cardiac damage. Similarly, in the absence of cardiac damage, myoglobin may be used as a biomarker of skeletal muscle injury.

PRINCIPLE OF THE ASSAY

The rat myoglobin SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 1) assay uses two different myoglobinspecific antibodies. An affinity purified polyclonal antibody is conjugated to horseradish peroxidase (HRP) and a myoglobin specific monoclonal antibody is conjugated to acridan, a chemiluminescent substrate. When HRP and acridan conjugated myoglobin antibodies bind to myoglobin they 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 antibodies not bound to myoglobin and distant from HRP at a molecular level produce no signal. This principle allows the development of a homogeneous assay that allows rapid determination of myoglobin concentration.

The HRP and acridan conjugated antibodies provided with the kit are mixed with standards and diluted samples in wells of the 96-well white SPARCL™ plate provided with the kit2. 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 myoglobin is proportional to luminescence and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Anti- myoglobin HRP stock Store ≤ -70°C

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

• Mouse myoglobin stock Store at -20°C

• Sample diluent; YD25-1, 25 ml

• Conjugate diluent; CSD10-1, 10 ml

• Trigger solution; TS11-1, 11 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.