Haptoglobin Kit ELISA

N° du produit ABIN956103

Le kit ELISA Haptoglobin (ABIN956103) Cheval est un kit ELISA Colorimetric conçu pour quantifier Cheval Haptoglobin.

Aperçu rapide pour Haptoglobin Kit ELISA (ABIN956103)

Antigène: Haptoglobin (HP)

Reactivité: Cheval

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Type d'échantillon: Plasma, Serum

Détail du produit Haptoglobin Kit ELISA

Analytical Method: Quantitative

Attributs du produit: The Horse Haptoglobin ELISA is based on a solid phase enzyme-linked immunosorbent assay (ELISA). The assay uses affinity purified anti-horse haptoglobin antibodies for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated anti-horse haptoglobin antibodies for detection. Serum or plasma is denatured and subsequently diluted. The diluted sample is incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 30 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.

Ingrédients: Anti-horse haptoglobin antibody coated microtiter plate with 96 wells (provided as 12 detachable strips of 8)
Enzyme Conjugate Reagent, 11 mL
Horse Haptoglobin Calibrator (lyophilized), containing 100 µg/mL horse haptoglobin when reconstituted as detailed on the vial label
Wash Buffer (20X), 50 mL
Denaturing buffer, 25 mL
Diluent (10X), 25 mL
TMB Reagent (One-Step), 11 mL
Stop Solution (1N HCl), 11 mL.

Matériel non inclus: 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
Plate reader with an optical density range of 0-4 at 450 nm
Graph paper (PC graphing software is optional)

Information d'application

Plaque: Pre-coated

Préparation de l'échantillon:

General Note: Haptoglobin is present in normal horse serum at a concentration of ~1 mg/mL. In order to obtain values within the range of the calibration curve, we suggest that samples be diluted 32,000 fold using the following procedure for each sample to be tested:
1. Dispense 197.5 µL of denaturing buffer and 997.5 µL of 1X diluent into separate tubes.
2. Pipette and mix 2.5 µL of the serum/plasma sample into the tube containing 197.5 µL of denaturing buffer. This provides an 80 fold diluted, denatured sample. Please note: the sample must be diluted at least 20-fold in the denaturing buffer.
3. Allow the samples to incubate in denaturing buffer for at least 10 minutes at room temperature.
4. Mix 2.5 µL of the 80 fold diluted sample with the 997.5 µL of 1X diluent in the second tube. This provides a 32,000 fold dilution of the sample.
5. Repeat this procedure for each sample to be tested.

Procédure de l'essai:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 µL of calibrators and diluted 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 30 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 5 minutes.

Calcul des résultats:

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 concentration 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 100-3.13 ng/mL calibration range when tested at a dilution of 32,000, samples should be re-diluted appropriately and re-tested. In the event that alternative dilutions are to be tested, prepare them by diluting from the denatured sample (prepare as described above) into 1X diluent. A minimum final dilution of 1,000 fold must be achieved, i.e., at least a 50 fold dilution form the 20 fold diluted denatured sample into 1X diluent.

Restrictions: For Research Use only

Stockage

Stock: -20 °C

Stockage commentaire: 1. For optimum stability store the lyophilized calibrator at or below -20°C when the ELISA kit is received.
2. The remainder of 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. Test kits will remain stable until the expiration date provided that the components are stored as described above.

Date de péremption: The expiry date is stated on the label.

Détail du antigène

Antigène: Haptoglobin (HP)

Autre désignation: Haptoglobin

Sujet: Haptoglobin is an acute phase protein that is elevated up to nine fold in horse serum as a result of inflammation and infection. Measurement of haptoglobin provides a convenient marker of inflammation and disease in horses.

Pathways: Transition Metal Ion Homeostasis