Creatinine Serum (Low Sample Volume) Kit

Catalog No. ABIN2866597

Product Details

Target: Creatinine (CR)

Reactivity: Human, Mouse, Rabbit, Rat, Sheep, Mammalian

Detection Method: Colorimetric

Application: Detection (D)

Purpose: The DetectX® Low Sample Volume Serum Creatinine kit is designed to quantitatively measure creatinine present in serum samples using a 384-well plate format to minimize sample usage.

Brand: DetectX®

Sample Type: Serum, Plasma

Components: Clear 384 well Plate Bag containing a 384-well plate. 1 each
Creatinine Standard Calibrated to NIST Standard Reference Material 100 mg/dL
creatinine solution in deionized water. 100 μL
Assay Diluent A special diluent for use in the serum kit. 12 mL
Allow to warm completely to room temperature prior to use.
DetectX® Creatinine reagent 28 mL

Material not included: Distilled or deionized water.
Appropriate manual or automated dispensing equipment for adding 15 or 60 μL of reagents to 384-well plates.
If you are not using an automated system then a repeater pipet for dispensing 60 μL of reagent is needed with disposable tips.
Colorimetric reader capable of reading 384-well microplate optical density at 490 nm.
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Application Notes: This assay has been validated for human, mouse, rabbit, rat and sheep serum and EDTA and heparin plasma samples.
The end user should evaluate recoveries of creatinine in other plasma and serum samples being used.
For measuring Creatinine in urine samples, please refer to our DetectX® Urinary Creatinine Detection kits, Catalog Number K002-H1 or K002-H5.
Hemolyzed or lipemic samples should not be used with this kit.
Hemolyzed samples have shown a decrease in creatinine concentration with increasing hemoglobin, whereas lipemic samples have been shown to yield artificially high creatinine concentrations.
Please see our Hemoglobin Detection kit, K013-H1 for details of a convenient method to measure Hb levels in whole blood.

Comment:

Sample values: Eleven serum samples from a variety of different species were tested in the assay.
Values ranged from 0.78 to 1.45 mg/dL with an average of 1.00 mg/dL.

Sample Volume: 15 μL

Protocol: Only 15 μL of serum or plasma is needed per well.
A creatinine standard, calibrated to a NIST creatinine standard, is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Standards or samples are pipetted into the clear microtiter plate.
An assay diluent is added to all standards, controls and samples.
The color generating reaction is initiated with the DetectX® Creatinine Reagent, which is pipetted into each well.
The assay utilizes a kinetic absorbance method to overcome interference by colored compounds in serum.
The absorbance of the colored product is read after 1 minute in a microtiter plate reader capable of measuring 490nm wavelength.
At 30 minutes the optical density is read again.
The concentration of creatinine is calculated using the delta of the optical density readings at 30 and 1 minute compared to the curve generated from the standards, or by using the Excel worksheet available for free download at our web site.
The Jaffe reaction used in this kit has been modified to read creatinine levels in serum.

Reagent Preparation:

Allow the kit reagents to come to room temperature for 30 minutes.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Standard Preparation Label glass test tubes #1 through #4.
Pipet 120 μL of water into tube #1 and 50 μL into tubes #2-#4.
Carefully add 5 μL of the Creatinine stock solution to tube #1 and vortex completely.
Take 50 μL of the creatinine solution in tube #1 and add it to tube #2 and vortex completely.
Repeat these serial dilutions for tubes #3 and #4.
The concentration of creatinine in tubes 1 through 4 will be 4, 2, 1 and 0.5 mg/dL.
Water is used as a sample blank of 0 mg/dL. use all Standards within 2 hours of preparation.
Std 1 Std 2 Std 3 Std 4 Water Volume (μL) 120 50 50 50 Addition Stock Std 1 Std 2 Std 3 Volume of Addition (μL) 5 50 50 50 final Conc (mg/dL) 4 2 1 0.5

Sample Preparation:

All samples should be centrifuged for 15 minutes at 14,000 rpm in an Eppendorf type centrifuge prior to running in the assay.

Assay Procedure:

We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine creatinine concentrations.
1. Use the plate layout sheet on the back page to aid in proper sample and standard identification.
2. Pipet 15 μL of samples, water as the blank, or standards into wells in the clear plate.
3. Add 15 μL of Assay Diluent to all wells used. Allow to warm completely to room temperature prior to use. Set a timer to read 30 minutes and ensure that the plate reader is set to read optical density at 490 nm.
4. Observe wells, checking for bubbles. If bubbles are present, tap the plate gently to remove prior to addition of Reagent.
5. Add 60 μL of the DetectX® Creatinine Reagent to each well using a repeater pipet or other pipetting equipment. Immediately start the timer after adding the Creatinine Reagent to the last well.
6. Incubate at room temperature.
7. At 1 minute, read the optical density generated from each well in a plate reader capable of reading at 490 nm.
8. At 30 minutes, again read the 490 nm optical density generated from each well in the plate reader. NOTE: If you do not use the whole plate, mark the wells that have been used. The unused wells can be used for further measurements. Do not wash wells to reuse.

Calculation of Results:

Subtract the average Optical Density of the standards at 1 minute from the average Optical Density of the standards at 30 minutes and plot the result (Average Delta OD) versus the creatinine concentration of the standards.
Generate a linear regression line and use the equation, y=mx+b (y=Average delta OD, x=Creatinine Concentration: m=slope and b= intercept) to calculate the concentrations in the unknown samples.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The Creatinine Reagent contains hazardous chemicals.
It contains a solution of basic picric acid in a stabilizing solution.
The solution should not come in contact with skin or eyes.
Picric acid is an irritant and, if dried, potentially explosive.
Avoid contact with metals and use large volumes of water during disposal.
Take appropriate precautions when handling these reagents.

Storage: 4 °C

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit.

References

Chin, Item, Wueest, Zhou, Wiedemann, Gai, Schoenle, Kullak-Ublick, Al-Hasani, Konrad: "Opposing effects of reduced kidney mass on liver and skeletal muscle insulin sensitivity in obese mice." in: Diabetes, Vol. 64, Issue 4, pp. 1131-41, (2015) (PubMed).

Target Details

Target: Creatinine (CR)

Alternative Name: Creatinine (CR Products)

Target Type: Amino Acid

Background: Creatinine (2-amino-1-methyl-5H-imadazol-4-one) is a metabolite of phosphocreatine (p-cre- atine), a molecule used as a store for high-energy phosphate that can be utilized by tissues for the production of ATP1. Creatine either comes from the diet or synthesized from the amino acids arginine, glycine, and methionine. This occurs in the kidneys and liver, although other organ sys- tems may be involved and species-specific differences may exist2. Creatine and p-creatine are converted non-enzymatically to the metabolite creatinine, which diffuses into the blood and is excreted by the kidneys. In vivo, this conversion appears to be irreversible and in vitro it is favored by higher temperatures and lower pH 2. Creatinine forms spontaneously from p-creatine3. Under normal conditions, its formation occurs at a rate that is relatively constant and as intra-individual variation is <15 % from day to day, creatinine is a useful tool for normalizing the levels of other molecules found in urine. Additionally altered creatinine levels may be associated with other con- ditions that result in decreased renal blood flow such as diabetes and cardiovascular disease4-6. 2- PO3 AtP ADP nH nH2 HOOC HOOC nH nH Creatine Kinase n n Creatine H Phosphocreatine O n nH n Creatinine