Rat ALPHA-1-Acid Glycoprotein SPARCL™ Assay | AGP-SP-2

BACKGROUND

Alpha-1-acid glycoprotein (AGP) is a heavily glycosylated protein that is expressed in the liver and secreted into blood. It is a positive acute phase reactant; liver expression and serum concentrations increase as a result of injury, infection or disease. In studies at Life Diagnostics, Inc. we found that serum AGP levels increased approximately 25-fold twenty-four hours after rats were challenged with lipopolysaccharide. AGP is an excellent acute phase biomarker in rats.

PRINCIPLE OF THE ASSAY

The rat AGP SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 1) assay uses two affinity purified AGP specific antibodies. One is conjugated to horseradish peroxidase (HRP), the other to acridan, a chemiluminescent substrate. When HRP and acridan conjugated AGP antibodies bind to AGP 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 distant from HRP produce no signal. This principle allows the development of a homogeneous assay that allows rapid determination of AGP concentrations.

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 AGP is proportional to luminescence and is derived from a standard curve.

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Anti-rat AGP HRP conjugate Store ≤ -70°C

• Anti-rat AGP acridan conjugate Store ≤ -70°C

• AGP stock 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

• Graphing software

STORAGE

Store the HRP conjugate, acridan conjugate and AGP stock at or below -70°C. 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 1 The SPARCL™ technology was developed by Lumigen Corp. 2 The white SPARCL™ plate provided with the kit has been treated with a reagent that reduces background chemiluminescence. Untreated plates cannot be used. antioxidant. The kit will remain stable for at least six months from the date of purchase, provided that the components are stored as described.

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.