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1. e Vortex sonicate Capture Beads Premix e Add 10 ul Capture Beads Premix to each well e Vortex mix plate 10 sec e Shake for 1 h 750 rpm room temperature in the dark Detection Antibody Incubation e Wash and vacuum plate 2X 100 ul Assay Buffer Type 2 e Add 40 ul Detection Antibodies mix to each well e Vortex mix plate 10 sec e Shake for 1 h 750 rpm room temperature in the dark e NOTE Do NOT wash or vacuum filter plate after incubation Streptavidin PE SA PE Incubation e Dilute 15X SA PE as needed For entire plate Add 144 ul 15X SA PE 2016 ul Assay Buffer Type 2 e Add 20 pl diluted 1X SA PE to each well e Shake for 30 min 750 rpm room temperature in the dark e Wash and vacuum plate 2X 100 ul Assay Buffer Type 2 e Resuspend beads in 100 ul Assay Buffer Type 2 Analysis e Shake approx 5 min 750 rpm room temperature in the dark Analyze on xMAP system e Low Gain RPI setting BioPlex e DD Gate 7500 15500 Luminex or default BioPlex e Sample size 50 pl e Collect 50 100 events per bead region e Timeout 60 sec USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk2. customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 8 of 9 Troubleshooting Problem Probable Cause Solution Leaking wells in filter plate Wicking due to adherent drops Tap filter plate several times on paper towel before adding samples or reagents Do not place filter plate on an absorbent surface during incubations If wells leaked during data acquisition it is possible to reacquire these wells Blot underside of wells and add 100 ul well Assay Buffer Type 2 Perforation of filter plate membranes Adjust the vacuum setting to lt 5 in 127 mm Hg Do not touch membranes with pipet tips Filter plate wells not draining under vacuum Vacuum is too low Adjust vacuum setting to 3 5 in 76 127 mm Hg Clean rubber seals Apply fingertip pressure to filter plate to ensure formation of a good seal Use the plate sealer to cover wells not in use Clogged membranes Clarify samples by centrifugation or filtration If samples are viscous dilute further before assaying Use the non tip end of a 200 ul pipette to flick the center support on the underside of the well then reapply vacuum Low bead counts during data acquisition No beads in the wells See Leaking wells in filter plate solutions above Verify that beads were added at the correct concentration and that correct bead regions and wells were selected during acquisition setup xMAP flui
3. 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 4 of 9 Rat Kidney Toxicity Panel 2 Protocol Considerations Before You Begin e Guidelines when using filter plates and vacuum manifold e Excessive vacuum will cause the filter plate membrane to perforate Adjust the pressure using a non filter ELISA or tissue culture plate ensuring that vacuum does not exceed 5 in 127 mm Hg e After adjusting the vacuum with a non filter plate place filter plate on the manifold Use fingertips to apply pressure evenly across the plate The liquid should drain in 2 5 sec e To avoid drying out the beads vacuum only long enough to drain all wells Do not allow drained beads to sit for gt 5 min before rehydrating with buffer e Itis critical to remove excess buffer from the underside of the filter plate by tapping it on a paper towel several times before adding samples or reagents This prevents samples from wicking out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations e To avoid perforating the filter plate membrane make certain that the probe height on the xMAP system is adjusted correctly Do not touch the membrane with pipet tips For accurate pipetting touch tips to the sides of the filter plate wells Change t
4. A 0109 Page 2 of 9 About the Kit WideScreen Rat Kidney Toxicity Panel 2 72174 3 Overview Bead based flow cytometric assays enable sensitive precise quantitation of analytes within a sample When directed toward proteins or peptides such assays are essentially ELISAs on a bead Samples are combined with fluorescently labeled microparticles beads covalently conjugated to a capture antibody Analytes captured on the beads are identified with detection antibodies and a fluorescent label A major advantage of bead based assays over traditional protein quantitation methods such as ELISA is the capacity for multiplexing as bead based assays allow simultaneous quantitation of multiple analytes in a small sample volume WideScreen Rat Kidney Toxicity Panel 2 is a pre mixed multiplex bead kit of antibody based assays for simultaneous quantitation of five kidney damage biomarkers in rat urine and plasma Analyte Full Alternate name Calbindin Calbindin Clusterin Clusterin Apolipoprotein J Cystatin C Cystatin C NGAL Neutrophil gelatinase associated lipocalin Osteopontin Osteopontin An essential aspect of drug development is determining detrimental side effects preferably prior to clinical trials The WideScreen Rat Kidney Toxicity Panels measure key biomarkers found in urine that can indicate drug induced damage to kidneys known as renal toxicity or nephrotoxicity Traditional tests of this nature test for biomarkers that are detect
5. Kidney Toxicity Panel 2 can also be used with rat plasma samples Please refer to sample preparation section on p 5 USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 3 of 9 Note Caution Components and Storage The kit includes all the reagents and buffers needed to assay the above proteins in rat urine samples using the Luminex xMAP System The kit contains sufficient components to assay one 96 well plate WideScreen Rat Kidney Toxicity Panel 2 72174 3 1 1 ml Rat Kidney Toxicity Panel 2 Capture Beads PBS with BSA Tween 20 and 0 025 ProClin 300 1 vial Rat Kidney Toxicity Panel 2 Detection Antibodies Lyophilized biotinylated detection antibody premix 1 vial Rat Kidney Toxicity Panel 2 Standards Mix Lyophilized recombinant protein standards for calbindin clusterin cystatin C NGAL and osteopontin 1 vial Rat Kidney Toxicity Panel 2 Control 1 Lyophilized low levels of calbindin clusterin cystatin C NGAL and osteopontin 1 vial Rat Kidney Toxicity Panel 2 Control 2 Lyophilized high levels of calbindin clusterin cystatin C NGAL and osteopontin 60 ml Assay Buffer Type 2 Store all 1X proprietary mix o
6. aS Novagen WideScreen Rat Kidney Toxicity Panel 2 Table of Contents RULES BASED MEDICINE About TNE KIE eogi a aaa a Oa Soe aaa oot 2 Overview 2 Components and Storage 3 Additional Materials Required But Not Supplied 3 Rat Kidney Toxicity Panel 2 ProtoCol 0 ccccccceceecsscsessescsseseessseeeeeees 4 Considerations Before You Begin 4 Reagent Preparation 4 Test Sample Preparation 5 Standard Dilution Series Preparation 5 Immunoassay Protocol 5 Collecting Data and Data Analysis ccccccscsssseeecscscsesssseseeesscsesaeees 7 Data Acquisition 7 Generation of Standard Curves and Quantitation of Experimental Samples 7 TrOUDIESHOOUNG s c siitiaid Antillana nied nial anidhini ase neianiaee 8 Appendix A Flowchart for Rat Kidney Toxicity Panel 2 Immunoassay 9 2009 EMD Chemicals Inc an Affiliate of Merck KGaA Darmstadt Germany All rights reserved The Novagen name and logo are registered trademarks of EMD Chemicals Inc in the United States and in certain other jurisdictions WideScreen is a trademark of EMD Chemicals Inc Bio Plex is a registered trademark and Bio Plex Manager is a trademark of Bio Rad Laboratories Inc Luminex and xMAP are registered trademarks and Luminex 100 IS is a trademark of Luminex Corporation ProClin is a registered trademark of Rohm and Haas Co Tween is a registered trademark of ICI Americas Inc Manufactured by Rules Based Medicine Inc By opening th
7. able days or weeks after kidney damage has occurred In contrast the WideScreen Rat Kidney Toxicity Panels provide researchers with a tool to detect biomarkers which may be upregulated and reveal damage within hours allowing drugs to be efficiently and rigorously tested before human clinical trials begin e Calbindin is a calcium binding protein found in epithelial cells involved in Ca transport including those in the cortical collecting tubules of the kidney e Clusterin is a highly conserved protein that is elevated following apoptotic cell death in a variety of tissues establishing it as a marker of apoptotic cell loss e Cystatin C is an extracellular inhibitor of cysteine proteases and is normally expressed in vascular smooth muscle cells Cystatin C levels correlate inversely with glomerular filtration rate e NGALis a protease resistant polypeptide detected in urine shortly following renal ischemia e Osteopontin promotes inflammation at sites of tissue injury and is upregulated following renal ischemia The WideScreen Rat Kidney Toxicity Panel 2 is a pre mixed multiplex bead kit of quantitative antibody based assays for simultaneous detection of five proteins found in rat urine and associated with kidney damage calbindin clusterin cystatin C NGAL and osteopontin The kit includes all the reagents and buffers needed to analyze the above proteins in rat urine samples using the Luminex xMAP System Note WideScreen Rat
8. d or lab tape for future use 2 Pre wet 96 well filter plate wells with 50 ul Assay Buffer Type 2 and incubate for a minimum of 5 min Immediately prior to Step 3 remove liquid from filter plate by vacuum filtration Do not exceed 5 in Hg or 127 mm Hg vacuum liquid should drain in 2 5 sec Tap filter plate on a paper towel to remove any buffer remaining on the underside Note It is critical to remove excess buffer from the underside of the filter plate before adding samples or reagents Otherwise samples may wick out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations If a well does not USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 6 of 9 drain use the non tip end of a 200 ul pipet tip to flick the center of the plastic support on the underside of the well then reapply vacuum 3 Add 10 ul of Blocking Buffer Type 4 to each filter plate well that will be used 4 Add 30 ul of each standard sample or control to appropriate well of the 96 well filter plate Note Rat Kidney Toxicity Panel 2 Control 1 and Control 2 do not need to be diluted 5 Vortex the plate by gently glidin
9. dics system is clogged Clear system of clogs or air using maintenance steps described in the instrument user manual sanitize alcohol flush probe sonication etc Make sure that the probe height is set correctly Make sure that beads are in suspension by incubating plate for 3 5 min on plate shaker 750 rpm immediately before analysis Microbial growth in buffers can cause beads to stick to the filter plate membrane Do not use contaminated reagents Timeout limit is set too low 50 100 events per bead region should be acquired within the 60 sec timeout limit If necessary the timeout limit can be set higher e g 75 sec Beads are not falling into the gates properly Beads were not resuspended in Assay Buffer Type 2 before analysis The setting of the Doublet Discriminator DD gate is buffer specific This gate can be adjusted but Assay Buffer Type 2 is the recommended buffer for running samples Other buffers may cause bead aggregation DD gate setting not optimal Use the DD gate setting recommended in the Data Acquisition Section If necessary raw data results can be reanalyzed with different DD gate settings see software user manual Beads were exposed to organic solvents Do not use organic solvents in the immunoassay as they will damage beads irreversibly Beads are falling outside the bead region gates due to photobleaching Do not use expired beads Do not expose the beads to ambient
10. e found in the Certificate of Analysis for the specific lot Refer to the Certificate of Analysis for expected control ranges The eight data points obtained with the concentration standards are plotted using Median Fluorescent Intensity MFI as the signal readout Y axis against concentration of standard dilutions X axis Five parameter logistic SPL curve fitting is recommended for modeling data obtained from bead based immunoassays Most ranges of standard concentrations are too wide for accurate linear regression analysis Four parameter logistic 4PL equations will often give a good fit but are not ideal because they assume the standard curve is symmetrical If the signal from an unknown sample exceeds the highest point of the standard curve the concentration of the unknown should not be calculated by extrapolation because the non linear shape of the standard curve cannot be accurately modeled past the last measured point In this case dilute the samples and test again When concentrations of unknowns have been determined by reading off of the standard curve remember to multiply this value by the dilution factor to obtain the concentration of the target in the original sample USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com
11. e packaging containing this product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this product for a full refund prior to using it in any manner You the buyer acquire the right under Luminex Corporation s patent rights if any to use the product or any portion of this product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument The terms and conditions governing EMD Chemicals sale of this product are as indicated on our website www emdbiosciences com USA and Canada Europe All Other Countries Tel 800 526 7319 France Germany Ireland United Kingdom All other Contact Your Local Distributor novatech novagen com Freephone Freecall Toll Free Freephone European Countries www novagen com 0800 126 461 0800 100 3496 1800 409 445 0800 622 935 44 115 943 0840 novatech novagen com techservice merckbio eu www novagen com FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE User Protocol TB523 Rev
12. el 2 can also be used with rat plasma samples The optimal dilution for plasma samples must be determined empirically However as a starting point we recommend a 5 fold dilution in Sample Dilution Buffer Type 3 For examples of dilutions used for plasma and urine samples see the Representative Data section of the Certificate of Analysis Standard Dilution Series Preparation This preparation provides sufficient volume to run duplicate standard dilution curves Label 8 polypropylene tubes S8 through S1 Alternatively prepare standard dilutions in a 96 well plate Pipet Standard Curve Diluent Type 5 into labeled tubes as described below Transfer the reconstituted Rat Kidney Toxicity Panel 2 Standards Mix to the S8 labeled tube Prepare 3 fold serial dilutions of S8 following the table below Ensure that each new standard is mixed well by vortexing before proceeding to the next dilution Change tips between each dilution Standard Volume of Standard Curve Diluent Volume of Standards Mix Type 5 S8 Oul 150 ul from vial S7 80 ul 40 ul of S8 S6 80 ul 40 ul of S7 S5 80 ul 40 ul of S6 S4 80 ul 40 ul of S5 83 80 ul 40 ul of S4 S2 80 ul 40 ul of S3 S1 80 ul 40 ul of S2 Note Standard concentrations are lot specific Refer to Certificate of Analysis of appropriate lot for specific standard concentrations Immunoassay Protocol 1 Seal any unused wells of the 96 well filter plate with plate sealer include
13. es USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de All Other Countries www novagen com novatech novagen com United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 9 of 9 Appendix A Flowchart for Rat Kidney Toxicity Panel 2 Immunoassay Pre wet Filter Plate e Add 50 ul Assay Buffer Type 2 to each well being used Prepare Reagents e Reconstitute all lyophilized reagents Standards Mix 150 ul dH20 Controls 1 and 2 100 ul dH 0 each Blocking Buffer 1 5 ml dH 0 Standard Curve Diluent 1 0 ml dH 0 Detection Antibodies 4 4 ml dH 0 Prepare Diluted Test Samples e Dilute urine 50 fold in Sample Dilution Buffer Type 3 e If further dilutions are needed perform in Sample Dilution Buffer Type 3 Prepare 8 point Standard Dilution Series Duplicates e 80 ul Standard Curve Diluent Type 5 in tubes S7 S1 e 150 ul Standards Mix in tube S8 e 3 fold serial dilutions mix thoroughly 40 ul from tube S8 to tube S7 etc Blocker Analyte Capture Bead Incubation e Remove liquid from pre wet filter plate by vacuum e Add 10 ul Blocking Buffer per well being used e Add 30 ul of the following and mix Test sample diluted or Controls 1 or 2 undiluted or Standard Dilution Series
14. f domestic animal proteins in PBS with components at 0 025 ProClin 300 4 C 1 vial Blocking Buffer Type 4 Lyophilized proprietary mix of domestic animal proteins to minimize non specific interactions 60 ml Sample Dilution Buffer Type 3 1X proprietary mix of domestic animal proteins in PBS with 0 025 ProClin 300 1 vial Standard Curve Diluent Type 5 Lyophilized proprietary mix of domestic animal proteins 150 ul 15X Streptavidin Phycoerythrin PBS with 2 mM NaN 1 96 well Filter Plate and Sealer Following reconstitution of lyophilized reagents store any unused reagent at 70 C See Reagent Preparation section p 4 WideScreen Rat Kidney Toxicity Panel 2 is not compatible with other bead kits and buffers sold by EMD or other vendors All materials derived from animal fluids or tissues should be considered biohazardous and handled accordingly Refer to MSDS for additional information Additional Materials Required But Not Supplied Luminex xMAP System or equivalent Vacuum manifold for filter plates Pall 5017 or Millipore MSVMHTSO0 96 well plate platform shaker such as IKA MTS4 Polypropylene microcentrifuge tubes 15 ml polypropylene centrifuge tubes Vortex mixer Ultrasonic bath such as Cole Parmer EW 08849 optional Multichannel pipet optional USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100
15. g the plate over the vortex mixer Note Gradually increase the vortex speed from low to medium Hold the plate with a loose grip Mix thoroughly for 10 sec Avoid splashing Alternatively mix using a plate shaker for 10 sec on high speed 1200 rpm 6 Sonicate 10 sec optional and vortex the tube of Rat Kidney Toxicity Panel 2 Capture Beads for 10 sec Add 10 ul to each well 7 Vortex or shake the plate 10 sec as described above in Step 5 Cover plate with aluminum foil to protect from light and incubate 1 hr at room temperature on a plate shaker 750 rpm 9 Remove liquid from filter plate by vacuum filtration 5 in Hg or 127 mm Hg maximum 10 Wash beads by adding 100 ul Assay Buffer Type 2 to each well and applying vacuum to remove buffer Repeat wash step for total of two washes in Assay Buffer Type 2 After the second wash and vacuum tap the filter plate on paper towels to remove any buffer remaining on the underside Note Do not resuspend beads in Assay Buffer Type 2 after second wash 11 Add 40 ul Rat Kidney Toxicity Panel 2 Detection Antibodies to each well Vortex or shake the plate as described in Step 5 12 Cover plate with aluminum foil to protect from light and incubate 1 h at room temperature on a plate shaker 750 rpm Note Do not wash beads after Detection Antibody incubation 13 Microcentrifuge 15X Streptavidin PE briefly 5 sec to ensure all material is in the bottom of the tube If using all 96 wells di
16. ips as necessary to prevent cross contamination e Capture Beads and Streptavidin PE are light sensitive To avoid photobleaching keep beads and Streptavidin PE in microcentrifuge tubes covered Cover filter plates with aluminum foil during incubation steps e To prevent fluorescent dye loss do not use organic solvents with capture beads Beads are incompatible with DMSO concentrations gt 1 e Many of the washing and reagent dispensing steps may be done with an 8 channel or 12 channel pipet manual or automatic For accurate results use calibrated single channel pipets for manipulation of standards and test samples e Test samples should be stored at 70 C prior to use Reagent Preparation 1 Resuspend each of the following lyophilized reagents in deionized water immediately prior to performing the assay Reagent dH O Volume Rat Kidney Toxicity Panel 2 Standards Mix 150 ul Rat Kidney Toxicity Panel 2 Control 1 100 ul Rat Kidney Toxicity Panel 2 Control 2 100 ul Blocking Buffer Type 4 1 5 ml Standard Curve Diluent Type 5 1 0 ml Rat Kidney Toxicity Panel 2 Detection Antibodies 4 4 ml 2 Mix each vial by vortexing at medium speed for 15 sec Incubate at room temperature for a minimum of 5 min not to exceed 30 min and repeat vortexing step Rat Kidney Toxicity Panel 2 Detection Antibodies can remain at room temperature for up to 2 hours Note Following reconstitution store any unused reagen
17. light for gt 10 min Avoid intense light Fluidics system is not running properly Confirm that the waste container is not full the sheath fluid is not empty and the SD fluidics module is turned on Check system calibration using approved calibration beads Verify correct system pressure Confirm that the system is free of air or particulate buildup Follow maintenance steps in the instrument user manual Insufficient volume of an immunoassay reagent Solutions were not prepared or used as described in protocol Briefly spin tubes to collect reagents that might be trapped in the tube cap Confirm correct buffer dilutions and use If additional Assay Buffer Type 2 is needed PBS can be used for the final bead resuspension step If additional 96 well filter plates are required we recommend Millipore Cat No MSBVN1210 If there is insufficient volume of 15X Streptavidin PE for your final experiment making a slightly more dilute working stock e g 20 fold instead of 15 fold will not adversely affect results Sample measurements not within the standard curve Dilution of sample is too low or too high If values are higher than the standard curve dilute samples further in Sample Dilution Buffer Type 3 and repeat assay Target concentration is below detection Verify that curve fitting at the lower end of the standard curve is accurate Not all urine and plasma samples contain detectable levels of all analyt
18. lute 15X Streptavidin PE to 1X by adding 144 ul concentrated Streptavidin PE to 2016 ul Assay Buffer Type 2 Note Do not dilute the whole vial of Streptavidin PE if the entire kit will not be used Dilute only what is needed based on the number of wells Allow 10 extra for pipetting error If there is an insufficient volume of 15X Streptavidin PE for your final experiment making a slightly more dilute working stock will not adversely affect results 14 Add 20 ul 1X Streptavidin PE to each well 15 Cover plate with aluminum foil to protect from light and incubate 30 min at room temperature on a plate shaker 750 rpm 16 Remove liquid from filter plate by vacuum filtration 17 Wash beads by adding 100 ul Assay Buffer Type 2 to each well and applying vacuum to remove buffer Repeat wash step for total of two washes in Assay Buffer Type 2 After second wash and vacuum tap filter plate on paper towels to remove any buffer remaining on the underside 18 Add 100 ul Assay Buffer Type 2 to each well 19 Cover plate to protect from light Incubate 3 5 min at room temperature on a plate shaker 750 rpm 20 Analyze using a Luminex instrument USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk Use
19. r Protocol TB523 Rev A 0109 Page 7 of 9 Collecting Data and Data Analysis Note Data Acquisition For detailed instructions on the operation of Luminex systems refer to the user guide for your specific instrument and software General recommendations are given below 1 Using your Luminex system software prepare a protocol for the assay you will run Enter information for each bead target and for the standards samples and controls Standard concentrations are lot specific Refer to Certificate of Analysis of appropriate lot for specific standard concentrations 2 Select the following bead regions Analyte Bead Region Analyte Bead Region Calbindin 62 NGAL 50 Clusterin 12 Osteopontin 52 Cystatin C 44 3 Acquire data using the system settings shown below Software Sample Events per Timeout Doublet CAL2 Gain Size Bead Region Discriminator Setting Luminex IS or 50 ul 50 100 60 sec 7500 15500 default equivalent Bio Plex Manager default 50 100 60 sec default RP1 low 50 ul 4335 10000 If the time spent acquiring samples needs to be reduced collect as few as 50 events per bead region Generation of Standard Curves and Quantitation of Experimental Samples Protein standards are supplied in the Rat Kidney Toxicity Panel 2 kit allowing for accurate quantitation using a titrated standard curve Representative standard curves and assay performance information can b
20. ts at 70 C Unused reagents can be stored at 70 C for up to one month Avoid multiple freeze thaw cycles USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB523 Rev A 0109 Page 5 of 9 Test Sample Preparation 1 Thaw and dilute samples within 1 h of use Remove any particulates by centrifugation or filtration Avoid multiple freeze thaw cycles 2 Dilute urine samples 50 fold in Sample Dilution Buffer Type 3 For preparing duplicate samples recommended mix 2 ul sample 98 ul Sample Dilution Buffer Type 3 Mix well and store on ice 3 Higher or lower urine sample dilutions may be required to ensure readings within the ranges of the assay standards and should be determined empirically While 1 50 is the recommended starting point for this multiplex panel dilutions as low as 1 10 are acceptable for quantifying low target levels and a dilution range of 1 1000 100 000 may be necessary for cystatin C depending upon the level of kidney damage Perform all sample dilutions in Sample Dilution Buffer Type 3 For higher dilutions a two step process is recommended for accuracy such as a 1 100 followed by a 1 100 10 000 final dilution Notes WideScreen Rat Kidney Toxicity Pan
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