Cow Haptoglobin SPARCL™ Assay | HAPT-SP-11

INTRODUCTION

Haptoglobin is an acute phase protein that is elevated in cow serum and milk because of injury, infection, and disease. In studies at Life Diagnostics, we found that serum haptoglobin levels range from ~0.1 µg/ml in healthy cows to greater than 4 mg/ml in sick cows. Levels were ~50 ng/ml in milk from healthy cows and greater than 2000 ng/ml in milk from cows with mastitis (SCC = 200-300 cells/ml). Haptoglobin therefore provides a useful biomarker of inflammation and disease in cows.

PRINCIPLE OF THE ASSAY

The cow haptoglobin SPARCL™1 (Spatial Proximity Analyte Reagent Capture Luminescence, ref 1) assay uses two cow haptoglobin-specific antibodies. One is conjugated to horseradish peroxidase (HRP), the other is conjugated to acridan, a chemiluminescent substrate. When the HRP and acridan conjugated antibodies bind to haptoglobin 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 measurement of haptoglobin concentrations.

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

MATERIALS AND COMPONENTS

Materials provided with the kit:

• Anti-haptoglobin HRP conjugate Store ≤ -70°C

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

• Haptoglobin stock (3 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°C for one week). The remainder of the kit should be stored at 4°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.