Haptoglobin ELISA Kit

Catalog No. ABIN956167

Monkey Haptoglobin ELISA Kit, Colorimetric assay for quantification of Monkey Haptoglobin.

Quick Overview for Haptoglobin ELISA Kit (ABIN956167)

Target: Haptoglobin (HP)

Reactivity: Monkey

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Sample Type: Plasma, Serum

Product Details Haptoglobin ELISA Kit

Analytical Method: Quantitative

Characteristics: The Monkey Haptoglobin ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses affinity purified anti-monkey haptoglobin antibodies for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-monkey haptoglobin antibodies for detection. The test sample is diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. This results in haptoglobin molecules being sandwiched between the immobilization and detection antibodies. The wells are then washed to remove unbound HRP-labeled antibodies and TMB Reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of Stop Solution, changing the color to yellow, and optical density is measured spectrophotometrically at 450 nm. The concentration of haptoglobin is proportional to the optical density of the test sample.

Components: Anti-monkey haptoglobin antibody coated microtiter plate with 96 wells (provided as 12 x 8-well strips)
Enzyme Conjugate Reagent, 11 mL
Monkey Haptoglobin Calibrator (lyophilized) Note: International import/export restrictions apply to monkey derived products. In order to avoid such restrictions the monkey haptoglobin calibrator supplied with this kit is of non-monkey origin. The calibration curve obtained with this material is identical to that obtained with monkey haptoglobin.
Wash Buffer (20X), 50 mL
Diluent (10X), 25 mL
TMB Reagent (One-Step), 11 mL
Stop Solution (1N HCl), 11 mL.

Material not included: Precision pipettes and tips
Distilled or de-ionized water
Polypropylene or glass tubes
Vortex mixer
Absorbent paper or paper towels
Micro-Plate incubator/shaker mixing speed of ~150 rpm
A microtiter plate reader capable of measuring absorbance at 450 nm, with a bandwidth of 10 nm or less and an OD range of 0-4 OD
Graph paper (PC graphing software is optional)

Application Details

Plate: Pre-coated

Sample Preparation:

General Note: Our studies find that haptoglobin is present in monkey serum at concentrations of 0.3 to 2 mg/mL. In order to obtain values within the range of the calibration curve, we suggest that samples initially be diluted 100,000 fold using the following procedure for each sample to be tested:
1. Dispense 998 µL and 497.5 µL of 1X diluent into separate tubes.
2. Pipette and mix 2 µL of the serum/plasma sample into the tube containing 998 µL of diluent. This provides a 500 fold diluted sample.
3. Mix 2.5 µL of the 500 fold diluted sample with the 497.5 µL of diluent in the second tube. This provides a 100,000 fold dilution of the sample.
4. Repeat this procedure for each sample to be tested.

Assay Procedure:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and samples into the wells (we recommend that samples be tested in duplicate).
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Remove the incubation mixture using either a plate washer or by flicking plate contents into an appropriate Bio-waste container.
   5. Wash and empty the microtiter wells 5 times with 1X wash solution. This may be performed using either a plate washer (400 µL/well) or with a squirt bottle. The entire wash procedure should be performed as quickly as possible.
   6. Strike the wells sharply onto adsorbent paper or paper towels to remove all residual droplets.
   7. Add 100 µL of enzyme conjugate reagent into each well.
   8. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   9. Wash as detailed in 4 to 5 above.
   10. Strike the wells sharply onto absorbent paper or paper towels to remove residual droplets.
   11. Dispense 100 µL of TMB Reagent into each well.
   12. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   13. Stop the reaction by adding 100 µL of Stop Solution to each well.
   14. Gently mix. It is important to make sure that all the blue color changes to yellow.
   15. Read the optical density at 450 nm with a microtiter plate reader within 15 minutes.

Calculation of Results:

1. Calculate the average absorbance values (A450) for each set of reference calibrators, and samples.
   2. Construct a calibration curve by plotting the mean absorbance obtained from each reference calibrator against its concentration in ng/mL on linear graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of haptoglobin in ng/mL from the calibration curve.
   4. Multiply the derived concentration by the dilution factor to determine the actual concentration of haptoglobin in the serum/plasma sample.
   5. PC graphing software may be used for the above steps.
   6. If the A450 values of samples fall outside the calibration curve when tested at the suggested dilution of 100,000, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: The kit should be stored at 4°C and the microtiter strips should be kept in a sealed bag with desiccant to minimize exposure to damp air. The kit will remain stable until the expiration date provided that the components are stored as described above.

Expiry Date: The expiry date is stated on the label.

Target Details for Haptoglobin

Target: Haptoglobin (HP)

Alternative Name: Haptoglobin

Background: Haptoglobin is a hemoglobin binding protein that is elevated in serum during the acute phase response. Studies have demonstrated that levels of haptoglobin are elevated approximately five fold in serum of monkeys undergoing veterinary treatment. Haptoglobin is a useful biomarker of tissue injury, inflammation and infection in monkeys.

Pathways: Transition Metal Ion Homeostasis