Urinary Creatinine Assay Kit

1. 96-well Microtiter Plate : Two 96-well strip plates.
2. Creatinine Standard : One 0.5 mL vial of a 100 mg/dL Creatinine solution.
3. Creatinine Reaction Buffer : One 30 mL bottle.
4. Acid Solution : One 10 mL bottle.

1. 1X PBS and deionized water
2. Sonicator or homogenizer for sample preparations
3. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
5. Spectrophotometric microplate reader capable of reading 490 nm

• Measures creatinine levels in urine samples
• Detection sensitivity limit of 300 μg/dl
• Creatinine standard included

• 1X Assay Diluent: Dilute the Assay Diluent 1:10 with deionized water. Mix to homogeneity. Store the 1X Assay Diluent at 4 °C up to six months.
• Creatinine Reaction Reagent: Combine 3 parts of the Creatinine Reaction Buffer with 1 part Picric Acid Solution (eg. For 100 assays, combine 15 mL of Creatinine Reaction Buffer with 5 mL of Picric Acid). Mix thoroughly. Store this Creatinine Reaction Reagent at room temperature for up to one week. Note: Picric Acid is a strong oxidizing agent and is toxic. Exercise caution and use safety measures while handling. Avoid contact with eyes, skin, and clothing. If contact is made, flush with copious amounts of water. Do not let the solution dry or crystalize as the dry powder is explosive. Use plenty of water to remove spilled picric acid. Preparation of Urine Samples The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design. A set of serial dilutions is recommended for samples to achieve optimal assay results and minimize possible interfering chromogens. Undiluted urine samples should be stored at -80 °C prior to performing the assay. Urine samples with visible particulates should be centrifuged or filtered prior to testing. A minimum 1:20 dilution of urine samples into deionized water is recommended to remove matrix interference and achieve optimal assay results. Diluted samples should be assayed within 2 hours of preparation. Note: High concentrations of ascorbic acid, uric acid, glucose, ketones and cephalosporin antibiotics may interfere with the assay causing falsely high values. Do not report the result from specimens with suspected interfering chromogens.

Each creatinine standard and urine sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 50 μL of the diluted creatinine standards or urine samples to the 96-well microtiter plate.
2. Add 200 μL of the prepared Creatinine Reaction Reagent to each well using either a multichannel pipette or a plate reader liquid handling system. Mix thoroughly and carefully so as not to create foaming in the well.
3. Incubate 30 minutes on an orbital shaker at room temperature.
4. Read the plate at 490 nm and record data. These are the initial absorbance values.
5. Add 50 μL of the Creatinine Quencher to samples and standard wells. Mix thoroughly. Incubate for 5-10 minutes on an orbital shaker. Reread the plate again at 490 nm and record data. These are the final absorbance values. Note: The difference in absorbance before and after adding the quencher is due to creatinine- picrate interactions. The Creatinine Quencher will permanently destroy the creatinine-picrate complex and any associated absorbance values. Ensure that data for all standards and samples are recorded prior to adding the quencher to wells.

1. Calculate the initial average absorbance (Ai) values for each standard and sample.
2. Calculate the final average absorbance (Af) values for each standard and sample.
3. Subtract the final absorbance from the initial absorbance for the corrected absorbance (Ac). (Ac) = (Ai) - (Af)
4. Plot the absorbance for the creatinine standards versus the concentration of the creatinine standards to determine the best curve. Data can be linearized with log paper or regression analysis software applications.
5. Use the equation obtained from the linear regression of the standard curve to calculate the creatinine concentration of samples. Remember to account for all dilution factors. See Figure 2 for a typical standard curve equation. (Ac) - y intercept Creatinine (mg/dL) = x sample dilution slope Note: Multiply the creatinine concentration in mg/dl by 88.4 to convert the values into μmol/L (SI Unit conversion). 7