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1 Code No.KAL-KK135 For research use only Urinary Creatinine ELISA Kit Creatinine is a metabolic waste product formed due to non-enzymatic conversion of creatine and phosphocreatine, the most part of which are found in muscle tissues. Creatinine diffuses into the blood and is excreted by kidneys into the urine. In normal condition, the excretion of creatinine is a relatively constant(1-2% of total creatine pool per day). The amount of creatinine produced is proportional to an individual s muscle mass. The urinary creatinine levels are commonly used as tool of normalization for other molecules in the urine or other tests. In addition, determination of urinary creatinine is also useful for detecting muscular and kidney disease, and estimation the extent of impairment renal function. Our kit is convenient to quantify amount of urinary creatinine by using ELISA method. This kit is only for research use, not for diagnosis. Highly sensitive and specific Strip type well, antigen pre-coated microplate Assay range: mg/dl Metabolizm of Creatinine In the kidneys Arginine + Glycine Ornithine + Guanidoacetic acid In the liver S-adenosyl-L-methionine + Guanidoacetic acid S-adenosyl-L-methionine + Creatine In muscle Creatine + ATP p-creatine + ADP Pi H2O H2O Creatinine p-creatinine Urinary Excretion 1/6

2 measurement principle Code No.KAL-KK Incubate with sample. = antigen for plates = HRP labelled anti Creatinine-specific antibody = Creatinine (sample or standard) antigen pre-coated plate 2. Remove unbound substances 3. Detect enzymatic reaction. Kit Contents (1) Antigen coated microtiter plate,96 wells 1 plate (2) Creatinine standard 250l 2 (3) Antibody diluent 20mL 1 (4) HRP- anti Creatinine antibody concentrate(100) 60l 1 (5) OPD (o-phenylendiamine) tablets 2 tab. (6) Substrate solution 30 ml 1 (7) Stop solution 15 ml 1 (8) Wash buffer concentrate (20) 30 ml 1 (9) Dilution plate 1 plate Equipments to be supplied by the user (1) A microplate reader (2) A micropipet (3) A microplate washer Assay Method (1) Preparation of working solution Wash solution Make sure that wash buffer concentrate does not contain any crystallized material prior to use. Working solution is prepared by dilution 30 ml of wash buffer concentrate with 570 ml of distilled deionized water. For convenience this solution can be kept at 2-8 up t o 14 days. Creatinine Standard Prepare 6 standards by serial dilution of Creatinine standard concentrate (20mg/dl ) as followings (mg/dl) Standard solution 20mg/dl (µl Deionized water µl HRP-anti Creatinine antibody (100) Dilute 40l of Anti Creatinine antibody concentrate (100) with 4 ml of Dilution solution for 96 well reaction. Diluted antibody should not be stored. 2/6

3 Code No.KAL-KK135 Coloring solution Add one OPD tablet to 13 ml of Substrate buffer to reconstitute the coloring solution just before use. This solution should not be stored. (2) Preparation of urine sample Collect urine in sampling tube on demand. After centrifiguration at 1500rpm for 5min, dilute the resulted supernatant 20 times with distilled deionized water. Measure the amount of creatinine in remaining diluted supernatant for compensation. Prepared urine sample should be kept below -80 if necessary. (3) Assay procedure Pre-reaction Prepare standard control wells containing 70L of anti Creatinine antibody solution and 70µL of 6 standards20,10,5,2.5,1.25,0.625 mg/dlin dilution plate. Likewise prepare experimental wells containing 70µL of anti Creatinine antibody solution and 70µL of prepared urinary sample in the same plate. After settlement, incubate at room temperature for 30 minutes. *Above reaction volumes can be applied for double measurements of primary reaction. If single measurement, reduce to 40µL of each solution. Preparation of reaction plate -1 Add wash solution 300l to each well and wait another 30 minutes. -2 Discard the wash solution from the wells completely and wash with 300l wash solution. Repeat this step another 2 times Primary reaction -1 Apply 50l/well2 (In the case of measuring double wells) pre-reaction solution(see ) and incubate for 1 hour. -2 After the incubation, discard the reaction solution and wash with 300l wash solution. Repeat this step another 2 times. Coloring Apply 100l Coloring solution to each well and incubate for 10 minutes at room temperature. Stop reaction Apply 100l of Stop solution to stop the enzymatic reaction Read absorbance Read absorbance of 490nm or 492 nm with a microplate reader. Measure concentration Measure the Creatinine concentration using standard curve. If actual measurements of sample exceed over 20mg/dl, dilute those urine samples again as possible as to evaluate within the range of mg/dl Concentration of Creatinine needs to be calculated from actual measurements by consideration of dilution ratio. 3/6

4 Standard curve Code No.KAL-KK OD Creatinine conc. (mg/dl Reproducibility Domain of standard curve : mg/dl Minimum measurement range for detection : 1.25mg/dl Minimun dilution number of urine sample : 20 Minimum sensitivity for detection : 25mg/dl Within-run (n=15, 2 concentration) : CV(%) = 8.40, 6.50 Between-run (n=10, 2 concentration) : CV(%) = 8.04, 7.41 Recovery test : In the recovery study, recoveries between 92% and 100% were obtained for 20, 40 times dilutions of the sample urine Coexistence substanceno influence to Hemoglobin 4500mg/dLBilirubin 180mg/dL Glucose 1000mg/dLAscorbic acid 500mg/dL Usage notes The Reagents should be stored at recommended temperature, -30. Do not use the reagents which is expired the date of usage. Urine sample should be diluted more than 20 times with Dilution solution. Do not leave the standard and Antibody for long time under room temperature. The glassware for making coloring solution should be clean. Since OPD (o-phenylendiamine) is harmful, handle with care. Since Stop solution,1n H2SO4, is strong acid, handle with care. The kit is constructed with well-adjusted combination in each lot. Replaced combination among different lots may cause unexpected results. This kit is only for research use. Do not use for medicinal or any other purposees. When using the reagents, take care to avoid them from touching to skin, mucous membrane, clothes, and getting into eye. If the reagents happen to get into eye or mouth, wash out them and consult a doctor if you need. After using the kit, wash your hand very carefully. If you find that the packages of the reagents are broken or something wrong, do not use them. When you store the reagents, make sure to avoid them from vaporizing, falling down. 4/6

5 Code No.KAL-KK135 After using the reagents, the packages should be discarded under the established rule. We do not guarantee the quality of the packages and accompaniments if not used according this direction. Storage All reagents: -30 5/6

6 Code No.KAL-KK135 References 1. Iyengar MR, Coleman DW, Butler TM. Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and creatinine under physiological conditions. J Biol Chem Jun 25;260(12): Furter R, Kaldis P, Furter-Graves EM, Schnyder T, Eppenberger HM, Wallimann T. Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli. Biochem J Dec 15;288 ( Pt 3): Wang ZM, Gallagher D, Nelson ME, Matthews DE, Heymsfield SB. Total-body skeletal muscle mass: evaluation of 24-h urinary creatinine excretion by computerized axial tomography. Am J Clin Nutr Jun;63(6): Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev Jul;80(3): Review. 5. Miller RC, Brindle E, Holman DJ, Shofer J, Klein NA, Soules MR, O'Connor KA. Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations. Clin Chem May;50(5): Epub 2004 Mar 11. This product is generated from GANP mice. Distributor TOYO 2CHOME, KOTO-KU, TOKYO, , JAPAN export@cosmobio.co.jp Phone : FAX : /6

7 Code Code No.KK135 Metabolizm of Creatinine In the kidneys Arginine + Glycine Ornithine + Guanidoacetic acid In the liver S-adenosyl-L-methionine + Guanidoacetic acid S-adenosyl-L-methionine + Creatine In muscle Creatine + ATP p-creatine + ADP Pi H2O H2O Creatinine p-creatinine Urinary Excretion 1/5

8 Code Code No.KK135 ELISA = = 2/5

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11 Code Code No.KK Iyengar MR, Coleman DW, Butler TM. Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and creatinine under physiological conditions. J Biol Chem Jun 25;260(12): Furter R, Kaldis P, Furter-Graves EM, Schnyder T, Eppenberger HM, Wallimann T. Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli. Biochem J Dec 15;288 ( Pt 3): Wang ZM, Gallagher D, Nelson ME, Matthews DE, Heymsfield SB. Total-body skeletal muscle mass: evaluation of 24-h urinary creatinine excretion by computerized axial tomography. Am J Clin Nutr Jun;63(6): Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev Jul;80(3): Review. 5. Miller RC, Brindle E, Holman DJ, Shofer J, Klein NA, Soules MR, O'Connor KA. Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations. Clin Chem May;50(5): Epub 2004 Mar 11. 5/5

12 Code Code No.KK135 Metabolizm of Creatinine In the kidneys Arginine + Glycine Ornithine + Guanidoacetic acid In the liver S-adenosyl-L-methionine + Guanidoacetic acid S-adenosyl-L-methionine + Creatine In muscle Creatine + ATP p-creatine + ADP Pi H2O H2O Creatinine p-creatinine Urinary Excretion 1/5

13 Code Code No.KK135 ELISA = = 2/5

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16 Code Code No.KK Iyengar MR, Coleman DW, Butler TM. Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and creatinine under physiological conditions. J Biol Chem Jun 25;260(12): Furter R, Kaldis P, Furter-Graves EM, Schnyder T, Eppenberger HM, Wallimann T. Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli. Biochem J Dec 15;288 ( Pt 3): Wang ZM, Gallagher D, Nelson ME, Matthews DE, Heymsfield SB. Total-body skeletal muscle mass: evaluation of 24-h urinary creatinine excretion by computerized axial tomography. Am J Clin Nutr Jun;63(6): Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev Jul;80(3): Review. 5. Miller RC, Brindle E, Holman DJ, Shofer J, Klein NA, Soules MR, O'Connor KA. Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations. Clin Chem May;50(5): Epub 2004 Mar 11. 5/5

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较