Uric Acid/Uricase Assay Kit
Contents
1. Product Manual Uric Acid Uricase Assay Kit Catalog Number STA 375 400 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction For humans and higher primates uric acid is the final oxidation end product of purine nucleotide metabolism The enzyme xanthine oxidase makes uric acid from xanthine and hypoxanthine which are derived from purines Although most animals can metabolize uric acid to the easily excreted product allantoin humans lack the necessary enzyme urate oxidase uricase due to two nonsense mutations in the uricase gene Uric acid is released in hypoxic conditions and 1s usually excreted in the urine via glomerular filtration Approximately 70 of daily uric acid disposal occurs via the kidneys Like ascorbic acid uric acid is a strong reducing agent electron donor and a potent antioxidant In humans over half of the antioxidant capacity of blood plasma is derived from uric acid High levels of uric acid have been linked to impaired renal function polycythemia leukemia as well as consumption of foods high in nucleoproteins Genetic and acquired influences such as obesity and alcohol consumption influence uric acid concentrations Hyperuricemia induces or accelerates the development of gout kidney stones hypertension metabolic syndrome and renal and cardiovascular disease Within 5 25 of humans impaired renal kidney2. 5 Watanabe S et al Hypertension 2002 40 355 360 g l A REN OE Recent product citation Leiba A et al 2015 Uric acid levels within the normal range predict increased risk of hypertension a cohort study Am J Hypertens American doi 10 1016 j jash 2015 05 010 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 02012 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing CELL BIOLABS INC br a J a i j
3. Uricase HN o O o A JL N N NH H H Fluorescence H O Allantoin CO Probe di Figure 1 Uric Acid Assay Principle Related Products pe eS debo xe I4 d 29 9 x STA 320 STA 340 STA 342 STA 344 STA 345 STA 347 STA 360 STA 374 STA 378 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation OxiSelect Superoxide Dismutase Activity Assay Kit OxiSelect Intracellular ROS Assay Kit Green Fluorescence OxiSelect Hydrogen peroxide Peroxidase Assay Kit Fluorometric OxiSelect ORAC Activity Assay OxiSelect In Vitro ROS RNS Assay Kit Green Fluorescence OxiSelect Total Antioxidant Capacity TAC Assay Kit OxiSelect Human KIM 1 ELISA Kit Creatinine Assay Kit 3 J F JAA W ig CELL BIOLABS INC _ Kit Components Box 1 shipped at room temperature 1 Uric Acid Standard Part No 236001 One 100 mg tube of powder 2 Fluorescence Probe Part No 237502 One 200 uL amber tube of a 10 mM solution in DMSO 3 HRP Part No 234402 One 100 uL tube of 100 U mL solution in glycerol 4 lOX Assay Buffer Part No 234403 One 25 mL bottle Box 2 shipped on blue ice packs 1 Uricase Part No 237503 One 80 uL amber tube of 100 U mL Note One unit is defined as the amount of enzyme that will oxidize 1 0 umole of uric acid to allantoin per minute at pH 8 5 and 25 C Materials Not Supplied per dw idb d de d i IN NaOH and deionized water 10
4. 0 mM Tris pH 7 5 1X PBS for sample dilutions and controls as necessary 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Standard 96 well fluorescence black microtiter plates Multichannel micropipette reservoir Fluorescence microplate reader capable of reading excitation in the 530 570 nm range and emission in the 590 600 nm range Storage Upon receipt aliquot and store the Fluorescence Probe HRP and Uricase at 20 C The Fluorescence Probe is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Store the remaining kit components at room temperature Preparation of Reagents Note All reagents must be brought to room temperature prior to use 1X Assay Buffer Warm the 10X Assay Buffer to room temperature prior to using Prepare a 1X Assay Buffer by diluting the 10X Assay Buffer with deionized water Add 25 mL 10X Assay Buffer to 225 mL deionized water for 250 mL total Mix to homogeneity Store the 1X Assay Buffer at 4 C up to 12 months Uric Acid Working Reagent Uric Acid Assay If measuring uric acid prepare a Uric Acid Working Reagent by diluting the Fluorescence Probe 1 100 HRP 1 250 and Uricase 1 250 in 1X 4 JN CELL BIOLABS INC Assay Buffer e g For 100 assays combine 50 uL of Fluorescence Probe 20 uL HRP and 20 uL Uricase with 1X Assay Buffer to a 5 mL total volume Mix thorough
5. ay Buffer Vortex thoroughly Prepare a series of the remaining uricase standards according to Table 2 below Prepare only enough for immediate use CELL BIOLABS INC i Fi 1 U mL Uricase Standard Standard 1X Assay Buffer Uricase mes uL uL mU mL lo 100 a 500 of Tube 1 3 500of Tube 2 LR L X 1 c s E 8 0 1 5 0 Table 2 Preparation of Uricase Standards Assay Protocol I Uric Acid II 1 Prepare and mix all reagents thoroughly before use Each sample including unknowns and standards should be assayed in duplicate or triplicate 2 Add 50 uL of each sample uric acid standards hydrogen peroxide controls or unknowns into an individual microtiter plate well 3 Add 50 uL of Uric Acid Working Reagent to each well Mix the well contents thoroughly and incubate for 20 minutes at 37 C and protected from light Note This assay is continuous not terminated and therefore may be measured at multiple time points to follow the kinetics of the reactions 4 Read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm range and for emission in the 590 600 nm range 5 Calculate the concentration of uric acid within samples by comparing the sample RFUs to the uric acid standard curve Subtract the value from the zero uric acid control well Uricase 1 Prepare and mix all reagents thoroughly before use Each sample including unknowns and standa
6. between 7 and for optimal working conditions as the Fluorescent Probe is unstable at high pH gt 5 Cell Lysate Resuspend cells at 1 2 x 10 cells mL in 1X Assay Buffer or PBS Homogenize or sonicate the cells on ice Centrifuge to remove debris Cell lysates can be assayed undiluted or titrated as necessary Serum Collect blood without using an anticoagulant and allow to clot for 30 minutes at 25 C Centrifuge at 2000 x g and 4 C for 15 minutes Remove the serum layer and store on ice Avoid disturbing the white buffy layer Aliquot samples for testing and store at 80 C Perform dilutions in 1X Assay Buffer or PBS Plasma Collect blood with heparin or citrate and centrifuge at 500 1000 x g and 4 C for 10 minutes Remove the plasma layer and store on ice Avoid disturbing the white buffy layer Aliquot samples for testing and store at 80 C Perform dilutions in 1 X Assay Buffer or PBS Urine To remove insoluble particles centrifuge at 10 000 rpm for 5 min The supernatant can be assayed directly or diluted as necessary Dilute in 1X Assay Buffer or PBS Aliquot samples for testing and store at 80 C Notes e All samples should be assayed immediately or stored at 80 C for up to 1 2 months Run proper controls as necessary Optimal dilution conditions for samples must be determined by the investigator Always run a standard curve with samples e Samples with NADH concentrations above 10 uM and glutathione concentrations above 50
7. excretion leads to hyperuricemia Gout is an inflammatory condition that results from uric acid deposits within the body joints Exercise induced acute renal failure or impairment is a major complication related to hypouricemia Cell Biolabs Uric Acid Uricase Assay Kit is a simple HTS compatible assay for measuring uric acid concentrations in biological samples such as serum plasma and urine without any need for pretreatment The kit has detection sensitivity limit of 0 5 uM of uric acid or 1 mU mL uricase Each kit provides sufficient reagents to perform up to 400 assays including standard curve and unknown samples Assay Principle The Uric Acid Uricase Assay Kit 1s a sensitive quantitative fluorometric assay for measuring uric acid or uricase concentrations Uric acid reacts with water and oxygen in the presence of the enzyme uricase to produce allantoin and H202 In the presence of HRP a Fluorescence Probe reacts with H202 in a 1 1 stoichiometry to produce a highly fluorescent product This fluorescent product can be easily read by a fluorescence microplate reader with an excitation of 530 560 nm and an emission of 590 nm Fluorescence values are proportional to the uric acid or uricase levels within the samples depending on which compound is being measured The uric acid or uricase content in unknown samples is determined by comparison with its respective standard curve Figure 1 Tha i E O N NH O A A H H O UricAcid O
8. ly and protect the solution from light For best results use the Uric Acid Working Reagent within 30 minutes of preparation Prepare only enough for immediate use Do not store the Working Reagent solution Uricase Working Reagent Uricase Assay Prior to measuring uricase activity weigh out the Uric Acid Standard powder for a 10 mg mL solution in IN NaOH This 10 mg mL is equivalent to a concentration of 60 mM Once dissolved dilute the 60 mM uric acid solution to a concentration of 10 mM in 100 mM Tris pH 7 5 Vortex thoroughly Next prepare the Uricase Working Reagent by diluting the Fluorescence Probe 1 100 HRP 1 250 and uric acid solution 1 10 in 1X Assay Buffer e g for 100 assays combine 50 uL of Fluorescence Probe 20 uL HRP and 500 uL Uric Acid with 1X Assay Buffer to a 5 mL total volume Mix thoroughly and protect the solution from light For best results use the Uricase Working Reagent within 30 minutes of preparation Prepare only enough for immediate use Do not store the Working Reagent solution Note If uric acid solution has visible precipitation or does not readily go into solution warm the solution at 37 C and vortex thoroughly to resuspend and dissolve Preparation of Samples Cell Culture Supernatant To remove insoluble particles centrifuge at 10 000 rpm for 5 min The supernatant can be assayed directly or diluted as necessary Prepare the standard curve in the same non conditioned media Note Maintain pH
9. rds should be assayed in duplicate or triplicate 2 Add 50 uL of each sample uricase standards hydrogen peroxide controls or unknowns into an individual microtiter plate well 3 Add 50 uL of Uricase Working Reagent to each well Mix the well contents thoroughly and incubate for 60 minutes at 37 C and protected from light Note This assay is continuous not terminated and therefore may be measured at multiple time points to follow the kinetics of the reactions 4 Read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm range and for emission in the 590 600 nm range 5 Calculate the concentration of uricase within samples by comparing the sample RFUs to the uricase standard curve Subtract the value from the zero uricase control well 1 CELL BIOLABS INC P i Fi Example of Results The following figures demonstrate typical Uric Acid Uricase Assay results One should use the data below for reference only This data should not be used to interpret actual results 20 40 Uric Acid uM Uric Acid uM Figure 2 Uric Acid Standard Curve 50 100 2 4 6 Uricase mU mL Uricase mU mL Figure 3 Uricase Standard Curve References 1 Heinig M and Johnson R J Cleve Clin J Med 2006 73 12 1059 1064 2 Tatyana V etal Neurochem 2001 79 266 3 Ichida K et al Genome Med 2009 1 12 118 4 Mahler H R et al J Biol Chem 1955 216 625 641
10. uM will oxidize the probe and could result in erroneous readings To minimize this interference it is recommended that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL Tayana et al Ref 2 5 Ja _ CELL BIOLABS INC LES ahe Solution e Avoid samples containing DTT or D mercaptoethanol since the probe is not stable in the presense of thiols above 10 uM e Uric acid values from urine can be normalized to creatinine levels Preparation of Standard Curves e Uric Acid Standards Prepare fresh Uric Acid standards by weighing out the Uric Acid Standard powder for a 10 mg mL solution in IN NaOH This 10 mg mL is equivalent to a concentration of 60 mM Use the 60 mM Uric Acid solution to prepare a 1 mM solution of Uric Acid eg add 50 uL of the 60 mM Uric Acid standard to 2 950 mL of 1X Assay Buffer Use the 1 mM solution to prepare a series of uric acid standards according to Table below Prepare only enough for immediate use Do not store the Working Reagent solution Note If uric acid solution has visible precipitation or does not readily go into solution warm the solution at 37 C and vortex thoroughly to resuspend and dissolve 1 mM Uric Acid Standard Standard 1X Assay Buffer Tubes uL uL Uric Acid uM 7 0 0 0 ee 0 Table 1 Preparation of Uric Acid Standards e Uricase Standard Prepare fresh uricase standards by first diluting the 100 U mL solution to 1 U mL in 1 X Ass
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