Urinary Creatinine Detection Kit

Sample values: 47 random clean catch human urine samples were tested in the assay.
Neat urine values ranged from 17.2 to 168.9 mg/dL with an average of 90.7 mg/dL.
One sample each of beagle and rat urines diluted 1:20 with water and read in the kit gave creatinine values in neat urine of 92.8 and 25.2 mg/dL respectively.
A single Rhesus monkey urine, diluted either 1:2 or 1:5, averaged 2.65 mg/dL in neat urine.

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 seven glass test tubes #1 through #7.
Pipet 800 μL of water into tube #1 and 500 μL into tubes #2-#7.
Carefully add 200 μL of the Creatinine Standard stock solution to tube #1 and vortex completely.
Take 500 μL of the creatinine solution in tube #1 and add it to tube #2 and vortex completely.
Add 500 μL of tube #2 to tube #3 and vortex completely.
Repeat these serial dilutions for tubes #4 through #7.
The concentration of creatinine in tubes 1 through 7 will be 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/dL.
Water will be used as a sample blank.
Use all standards within 2 hours of preparation.

Rhesus monkey urine samples contain very low levels of creatinine and should be diluted 1:2 in water by taking one part of urine and adding to one part of water prior to using in the assay. All other urine samples must be diluted 1:20 with deionized or distilled water by taking one part of urine and adding to nineteen parts of water to obtain accurate results. Any urine samples with creatinine concentrations outside the standard curve range should be diluted further with water to obtain readings within the standard curve. Use all diluted samples within 2 hours of preparation.

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 50 μL of samples, water as the blank, or standards into wells in the clear plate.
3. Add 100 μL of the DetectX® Creatinine Reagent to each well using a repeater pipet.
4. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and press to seal adequately.
5. Incubate at room temperature for 30 minutes.
6. Read the optical density generated from each well in a plate reader capable of reading at 490nm.
7. Use the plate reader's built-in 4PLC software capabilities to calculate creatinine concentrations for each sample.

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit, after subtracting the mean OD's for the blank.
The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.