CST3 Kit ELISA

N° du produit ABIN956265

Le kit ELISA CST3 (ABIN956265) Singe est un kit ELISA Colorimetric conçu pour quantifier Singe CST3.

Aperçu rapide pour CST3 Kit ELISA (ABIN956265)

Antigène: CST3 (Cystatin C (CST3))

Reactivité: Singe

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Type d'échantillon: Serum, Urine

Détail du produit CST3 Kit ELISA

Analytical Method: Quantitative

Attributs du produit: Test samples are diluted and incubated in microtiter wells for 45 minutes alongside prepared monkey Cystatin C calibrators. The microtiter wells are subsequently washed and HRP conjugate is added and incubated for 45 minutes. Cystatin C molecules are thus sandwiched between 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 Cystatin C is proportional to the optical density of the test sample and is derived from a calibration curve.

Ingrédients: Anti Cystatin C coated 96-well plate (12 strips of 8 wells)
Reference calibrator (lyophilized)
Diluent, 50 mL
HRP Conjugate Reagent, 11 mL
20X Wash Solution, 50 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
Vortex mixer
Absorbent paper or paper towels
Graph paper (PC graphing software is optional)
Polypropylene or glass tubes
Plate reader with an optical density range of 0-4 at 450 nm.
Micro-Plate incubator/shaker mixing speed of ~150 rpm
Plate washer

Information d'application

Plaque: Pre-coated

Préparation de l'échantillon:

During validation studies, monkey serum was found to have a Cystatin C concentration of ~1 µg/mL. We therefore suggest an initial serum dilution of 1,000-fold. This can be achieved with minimal use of diluent by first diluting 1 µL of serum with 99 µL of diluent to give a 100-fold dilution and then diluting 50 µL of the 100-fold diluted sample with 450 µL of diluent to give the 1,000-fold diluted sample. Normal urine samples were found to have Cystatin C concentrations of ~50 ng/mL and were diluted 50-fold with diluent prior to assay. In order to eliminate matrix effects, a minimum dilution of 25-fold for urine samples is recommended.
PROCEDURAL NOTES
1. Calibrators should be used within 30 minutes of preparation.
2. Optimum results are achieved if, at each step, reagents are pipetted into the wells of the microtiter plate within 5 minutes.
3. We recommend that calibrators and samples be run in duplicate.
4. The optical density of the microtiter wells should be read within 5 minutes following the addition of Stop Solution.

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.
   3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1x wash solution using a plate washer (400 µL/well). The entire wash procedure should be performed as quickly as possible.
   5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer.
   6. Add 100 µL of enzyme conjugate reagent into each well.
   7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 45 minutes.
   8. Wash as detailed in 4 to 5 above.
   9. Dispense 100 µL of TMB Reagent into each well.
   10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (18-25°C) for 20 minutes.
   11. Stop the reaction by adding 100 µL of Stop Solution to each well.
   12. Gently mix. It is important to make sure that all the blue color changes to yellow.
   13. 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 concentrations on the horizontal or X-axis.
   3. Using the mean absorbance value for each sample, determine the corresponding concentration of Cystatin C in ng/mL from the calibration curve.
   4. Multiply the derived concentrations by the dilution factor to determine the actual concentration of Cystatin C in the sample.
   5. PC graphing software may be used for the above steps.
   6. If the OD450 values of the samples fall outside the calibration curve, samples should be diluted appropriately and re-tested.

Restrictions: For Research Use only

Stockage

Stock: 4 °C

Stockage commentaire: The test kit will remain stable until the expiration date provided that the components are stored at 4°C. The microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air.

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

Détail du antigène

Antigène: CST3 (Cystatin C (CST3))

Autre désignation: Cystatin C

Sujet: Cystatin C is a cysteine protease inhibitor with a molecular weight of 13 kDa that is found in most body fluids. It is normally removed from blood by glomerular filtration in the kidneys, reabsorbed by the tubules and subsequently degraded. Acute kidney injury impairs this process decreasing both glomerular filtration and tubule function. The result is an increase in both serum and urine Cystatin C levels. Cystatin C is thus a useful biomarker of kidney injury.