Urinary Creatinine Detection Kit

Transcription

1 B-Bridge International, Inc. Urinary Creatinine Detection Kit User Manual Catalog # K Plate Kit K Plate Kit 1

2 Table of Contents Intended Use Background 3 Assay Principle Kit Components....4 Materials Required But Not Supplied...4 Precautions...4 Reagent Preparation....5 Sample Preparation.. 5 Assay Protocol Calculations....5 Typical Standard Curve....6 Notice to Purchaser This product is to be used for Research Purposes Only. It is not to be used for Drug or Diagnostic Purposes, nor is it intended for Human Use. B-Bridge products may not be resold, modified for resale, or used to manufacture commercial products without the express written consent of B-Bridge International, Inc. EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL, B-BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND, EITHER EXPRESSED OR IMPLIED, WITH RESPECT TO THE PRODUCTS, OR INFORMATION DISCLOSED HEREUNDER, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY, FIT FOR A PARTICULAR PURPOSE, OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES. B-Bridge International, Inc. All Rights Reserved. 2

3 Intended Use The B-Bridge Urinary Creatinine Detection Kit quantitatively measures creatinine present in urine. This assay is species independent and has been pre-validated for use with human, rat, dog, and monkey urine samples. Note: Mouse urine samples are not compatible with the use of this assay to determine Glomerular Filtration Rate (GFR) as over half of murine urinary creatinine is from renal secretion rather than filtration. Background Creatinine (2-amino-1-methyl-5H-imadazol-4-one) is a metabolite of phosphocreatine (p-creatine or PCr), a molecule used as a store for high-energy phosphate that can be utilized by tissues for the production of ATP. Creatine either comes from the diet or is synthesized from the amino acids arginine, glycine, and methionine. This occurs in the kidneys and liver, although other organ systems may be involved and species-specific differences may exist. Creatine and p-creatine are converted non-enzymatically to the metabolite creatinine, which diffuses into the bloodsteam and is excreted by the kidneys. In vivo, this conversion appears to be irreversible; in vitro the forward reaction is favored by high temperatures and low ph. As shown below, creatinine is spontaneously formed from p-creatine. Under normal conditions, its formation occurs at a rate that is relatively constant with intra-individual variation <15% from day to day. This makes creatinine a useful tool for normalizing the levels of other molecules found in urine. Additionally, altered creatinine levels may be associated with other conditions that result in decreased renal blood flow such as diabetes and cardiovascular disease. Assay Principle The Urinary Creatinine Detection kit is designed to quantitatively measure creatinine present in urine samples. Please read the complete kit insert before performing this assay. A colorimetric reaction is initiated with the Creatinine Detection Reagent in a modified Jaffe reaction; the concentration of creatinine present is determined from color intensity measured by a plate reader at a 490 nm wavelength. 1. Sample or standard added to well in clear microtiter plate. 2. A colorimetric reaction is initiated with the addition of the Creatinine Detection Reagent. 3. Incubate at room temperature for 30 minutes and read optical density with plate reader at 490 nm. 4. Calculate creatinine concentration from standard curve. 3

4 Kit Components Component: Cat # K K Clear 96-well microtiter plates 2 plates 10 plates Creatinine Standard (100 mg/dl in dionized water) 1 ml 2 x 1 ml - Calibrated to NIST Standard Reference Material Creatinine Detection Reagent 20 ml 2 x 50 ml Plate Sealers 2 each 10 each Store all components at 4 C Materials Required But Not Supplied Deionized or distilled water Colorimetric 96-well microplate reader capable of reading optical density at 490 nm, preferably with correction between 570 and 590 nm Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting. Contact your plate reader manufacturer for details Precautions As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction. The complete User Manual should be read and understood before attempting to use the product. The Creatinine Detection 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. In all cases, please consult your institution s safety procedures for working with hazardous chemicals. 4

5 Reagent Preparation Allow the kit reagents to come to room temperature for 30 minutes. We recommend that all standards and samples be run in duplicate to accurately determine creatinine concentrations. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to assaying. Standard Preparation Creatinine Standards are prepared by labeling seven test tubes as #1 through #7. Pipette 800 µl of distilled water into tube #1 and 500 µl into tubes #2-#7. Add 200 µl of the Creatinine Standard Stock Solution to tube #1 and vortex completely. Take 500 µl of the formaldehyde standard solution in tube #1 and add it to tube #2 and vortex completely. Repeat this serial dilution for tubes #3-#7, adding 500 µl of the preceding standard into each subsequent tube. The concentration of formaldehyde in tubes #1-7 will be 20, 10, 5, 2.5, 1.25, 0.625, mg/dl, respectively. Distilled water will be used as a sample blank. Use all standards within 2 hours of preparation. Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Distilled Water 800 µl 500 µl 500 µl 500 µl 500 µl 500 µl 500 µl Standard 200 µl Stock 500 µl Std µl Std µl Std µl Std µl Std µl Std 6 Concentration 20 mg/dl 10 mg/dl 5 mg/dl 2.5 mg/dl 1.25 mg/dl mg/dl mg/dl Sample Preparation Rhesus monkey urine samples contain very low levels of creatinine and should be diluted 1:2 in dionized or distilled water by taking one part of urine and adding one part of water prior to using the assay. All other urine samples should be diluted 1:20 with deionized or distilled water by taking one part of urine and adding 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. Assay Protocol 1. Pipet 50 µl of samples, standards, and distilled water as blanks in duplicate into wells in the clear microplate. 2. Add 100 µl of Creatinine Detection Reagent to each well using a repeater or multi-channel pipette. 3. Thoroughly mix reagents; cover plate with plate sealer and press to seal securely. 4. Incubate at room temperature for 30 minutes. 5. Read the optical density (OD) of each well in a plate reader at 490 nm wavelength. 6. Use the plate reader s built-in 4PLC software capabilities to calculate creatinine concentrations for each sample. Calculations Average the duplicate optical density (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) 5

6 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. Typical Standard Curve 6