TECO® Haptoglobin (Equine)

Materials Required and not Supplied

• Pipette capable of accurately dispensing 10-100μl and 100-1000μl
• Multichannel pipette for 100μl
• Graduated cylinders for reconstituting or diluting reagents
• Automatic washer or equivalent plate washing system
• Distilled or deionized water
• Vortex mixer
• ELISA plate shaker (orbital shaker, 500rpm)
• ELISA plate reader suitable for 96 well formats and capable of measuring at 450nm (Reference Filter: 590-650nm).
• ELISA plate reader software for data generation and analysis

ASSAY PRINCIPLE

The TECO® Equine haptoglobin EIA Kit is a 96 well sandwich ELISA product. The assay uses affinity purified anti-horse haptoglobin antibodies immobilized at solid phase (microtiter wells). Pre-diluted samples are incubated in the microtiter wells for 60min. The microtiter wells are subsequently washed, and horseradish peroxidase (HRP) conjugated anti-haptoglobin antibodies are added and incubated for 30 minutes for detection. After incubation, the unbound HAP-labeled antibodies are washed away.

TMB substrate is added which reacts with the HRP and resulting in a concentration-depended color level. The reaction is stopped with HCI changing the blue color to yellow, and the plate is read using a plate reader at 450nm.

Color development is proportional to the amount of haptoglobin in the sample.

ASSAY PROCEDURE

It is recommended that all determinations (Standards and samples) are assayed in duplicate. When performing the assay, Standards and samples should be pipetted as fast as possible (< 15 minutes).

To avoid distortions due to differences in incubation times, Substrate Solution and Stop Solution should be added to the plate in the same order and with the same time interval. Before use, allow all reagents to stand at room temperature (20-25°C) for at least 30 minutes. During all incubation steps, plates should be sealed with the adhesive foil or a plastic cover. For light protection, incubate in a dark chamber or cover plate with aluminum foil.

1. Allocate the wells of the Microwell Plate for Standards and samples
2. Pre-wash the microassay strips as follows:
   a. Using a wash bottle or automated plate washing device, add approximately 350μl Wash Buffer to each well.
   b. Incubate the wells for two minutes at 20-25°C.
   c. Aspirate the contents from each well. Invert the plate and tap firmly on absorbent paper to remove any remaining liquid.
3. Add 100μl Assay Buffer to each well (multichannel pipette)
4. Pipette 20μl of each Standard (  A  till  F  ) and diluted samples into the corresponding wells.
5. Incubate the plate for 1h at room temperature (20-25°C) on a shaker (500rpm).
6. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 times with 350μl diluted Wash Buffer per well. After the last wash cycle tap the inverted wells on a dry absorbent surface to remove excess wash solution. The use of an automatic plate washer is recommended.
7. Add 100μl of HRP Antibody Conjugate (multichannel pipette).
8. Incubate the plate for 30min in the dark at room temperature (20-25°C) on a shaker (500rpm).
9. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 times with 350μl diluted Wash Buffer per well. After the last wash cycle tap the inverted wells on a dry absorbent surface to remove excess wash solution. The use of an automatic plate washer is recommended.
10. Pipette 100μl of the TMB Substrate Solution in each well (multichannel pipette).
11. Incubate the plate for 15-30min in the dark at room temperature (20-25°C) on a shaker (500rpm).
12. Stop the reaction by adding 100μl of Stop Solution (multichannel pipette).
13. Measure the color reaction within 10min at 450nm (reference filter between 590-650nm).

Example only, not for use in calculation of actual results

Haptoglobin is an acute phase protein and is part of immune system-mediated defense mechanisms found in the blood various animal species. Under normal conditions, haptoglobin is either absent from the blood or present at very low levels. However, haptoglobin can increase significantly in response to acute infection, inflammation or trauma.