Description
Creatinine Assay Kit (BA0231) (BA0231)
The Creatinine Assay Kit (SKU: BA0231) provides a fast, sensitive enzyme-based method for determining creatinine in urine, serum, plasma and other biological samples. Creatinine is synthesised in the body from creatine, removed from the blood by filtration through the glomeruli of the kidney and secreted into urine, and in healthy individuals its secretion is largely independent of diet and fairly constant. The creatinine clearance test is one of the most sensitive measures of glomerular filtration rate: in kidney disease blood creatinine rises while clearance and urine levels fall, making creatinine testing widely used to assess kidney function. This assay uses a reaction sequence that excludes endogenous ammonia to convert a dye into a coloured and fluorescent form, with the absorbance at 570 nm or fluorescence at lambda ex/em = 530/585 nm directly proportional to the creatinine concentration. The procedure involves adding a single working reagent and reading after 60 minutes at room temperature, with no 37°C heater required, and can be readily automated for high-throughput use.
| Product Name: | Creatinine Assay Kit (BA0231) |
| SKU: | BA0231 |
| Detection Method: | Enzyme-based colorimetric (570 nm) or fluorimetric (lambda ex/em = 530/585 nm) detection. A reaction sequence that excludes endogenous ammonia converts a dye into a coloured and fluorescent form; the signal is directly proportional to the creatinine concentration in the sample. |
| Detection Range: | Linear detection range 4.8 to 500 µM (0.054-5.7 mg/dL) for the colorimetric assay and 0.25 to 100 µM (0.0028-1.14 mg/dL) for the fluorimetric assay, for a 60-minute reaction |
| Sample Type: | Urine, serum, plasma and other biological preparations |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This assay determines creatinine using creatininase and creatinase reactions in a sequence that excludes endogenous ammonia, converting a dye into a coloured and fluorescent form. The absorbance at 570 nm (colorimetric) or fluorescence at lambda ex/em = 530/585 nm (fluorimetric) is directly proportional to the creatinine concentration in the sample and is quantified against a standard curve.
- Fast and sensitive. Linear detection range 4.8 to 500 µM (colorimetric) and 0.25 to 100 µM (fluorimetric) for a 60-minute reaction.
- Convenient. Single working reagent, read after 60 minutes; room temperature assay with no 37°C heater required.
- High-throughput. Homogeneous mix-incubate-measure assay that can be readily automated for thousands of samples per day.
- Creatinine determination in urine, serum, plasma and other biological preparations.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Sample preparation: because the assay is based on creatinase and creatininase reactions, creatine in the sample can interfere. Dilute urine samples at least 100-fold with dH2O. Deproteinate serum and plasma by centrifugation for 15 minutes at 14,000 rpm at room temperature through a 10 kDa spin filter and assay the filtrate directly. |
| 2 | Reagent preparation: equilibrate all reagents to room temperature and briefly centrifuge tubes before opening. |
| 3 | Colorimetric standards: prepare 200 µL of 500 µM premix by mixing 50 µL of the Standard (2 mM) with 150 µL dH2O, then dilute to give standards of 500, 300, 150 and 0 µM. Transfer 20 µL of standards and 20 µL of samples into separate wells of a clear flat-bottom 96-well plate. |
| 4 | Reconstitute Enzyme A with 120 µL Assay Buffer. Prepare Working Reagent per well by mixing 82 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B and 1 µL Dye Reagent. Add 80 µL Working Reagent to each well and tap briefly to mix. |
| 5 | Incubate at room temperature for 60 minutes and read OD at 570 nm. |
| 6 | Fluorimetric procedure: dilute the colorimetric standards 1:5 in dH2O, transfer 20 µL standards and 20 µL samples into a black 96-well plate, add 80 µL Working Reagent, tap to mix, incubate protected from light for 60 minutes at room temperature and read fluorescence at lambda ex/em = 530/585 nm. |
Subtract the blank value (water, Standard #4) from the standard values and plot the adjusted values against standard concentrations to determine the slope. [Creatinine] = (R_SAMPLE - R_BLANK) / Slope (per µM) x n µM, where R_SAMPLE and R_BLANK are the OD or fluorescence values of the sample and water blank and n is the dilution factor. Conversion: 1 µM creatinine equals 0.0113 mg/dL.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20°C |
| Enzyme A | Dried | -20°C |
| Enzyme B | 120 µL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Standard | 1 mL | -20°C |
