xMAP® Antibody Coupling Kit User Manual
Contents
1. Protein Considerations Kit Contents Storage and Stability Necessary Materials and Equipment Instructions General Workflow Coupling Protocol Coupling Assessment Microsphere Enumeration Coupling Confirmation Sample Coupling Confirmation Protocol A Troubleshooting esseecsececseseecseceesseeeeeecsessesuesussessueeeeeteatenens Safety The following informational notes and cautions are used in this manual NOTE This message is used to provide general helpful information IMPORTANT This message advises users that failure to take action or avoid a certain action can result in data loss or the reporting of incorrect data You may encounter the symbols in the table below within this manual These represent warnings conditions identifications and important information Table 1 Symbols Symbol Meaning General Warning Caution Risk of Danger ud Manufacturer Decision Point Page 1 of 20 Material Safety Data Sheets MSDS s are available upon request Visit http www luminexcorp com support for more information Page 2 of 20 CAUTION xMAP Antibody Coupling Kit Sulfo NHS Solution MAY BE HARMFUL IF ABSORBED THROUGH SKIN OR IF SWALLOWED MAY CAUSE EYE AND SKIN IRRITATION CAUTION xMAP Antibody Coupling Kit Activation Buffer MAY BE HARMFUL IF ABSORBED THROUGH SKIN OR IF SWALLOWED CAUTION EDC causes severe eye irritation Causes respirat2. e Coupling optimization and assay optimization For users developing a new assay the kit will allow them to perform up to 10 small scale coupling reactions to test multiple concentrations of antibody with multiple bead regions e Small to medium scale assay manufacturing For users who have already developed and optimized an assay the kit will allow them to couple up to 50 million beads at once to meet their routine usage needs Page 3 of 20 With Luminex MagPlex microspheres if the kit protocol is performed carefully the percent recovery of the coupling reaction is typically 90 or greater enough for more than forty five 96 well plates 2 500 beads well for every 12 5x106 microspheres coupled Method Carboxylated microsphere 9 cr 9 o NH oslo fw a 7e ot we 0 N ji A o gt stable stable hi di amide bonc Unstable reactive w ane reactive Q IHS ester N o acylisourea ester 4 e EDC u Fr Sulfo NHS NOTE The diagram above illustrates the chemical reaction taking place during coupling and is not intended to be a literal representation of the order in which reagents are added to the reaction The coupling procedure involves a two step carbodiimide reaction The carboxyl groups on the surface of the polystyrene microspheres must first be activated with a carbodiimide derivative prior to coupling the antibody EDC 1 Ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride reacts with the carboxy
3. e Vortexer e Pipettor Optional Equipment e Luminex Tube Magnetic Separator Cat CN 0288 01 Microcentrifuge e Luminex Plate Magnetic Separator Cat CN 0269 01 Page 7 of 20 Instructions General Workflow Add Wash Add Incubate microspheres to microspheres _ sulfo NHS and EDC for reaction tube wl activation buffer solutions 20 mins Wash al Incubate Wash and resuspend microspheres a for microspheres wl activation buffer antibody or protein 2hrs wi wash buffer Sa Coupling Protocol The following is the detailed step by step process for coupling antibodies or similar proteins to carboxylated magnetic microspheres 8 IMPORTANT Protect photosensitive microspheres from light whenever possible throughout this entire procedure 1 Remove kit and all reagents from the refrigerator and allow them to equilibrate to room temperature for 20 30 minutes 2 While the reagents equilibrate to room temperature calculate and note the required volumes for Decision Point steps 4 8 11 13 19 and 20 for each reaction being performed 3 Resuspend the stock microspheres e If using a 1mL stock microsphere vial vortex the stock microsphere vial for 10 seconds and then sonicate for 10 seconds to disperse the microspheres Alternatively the microsphere vials can be rotated on a rotator for 15 minutes e If using a 4mL stock microsphere vial rotate the vial for 15 minut
4. in the pouch it is been stored correctly or rehydrated long before use shipped in until ready to use EDC must be used immediately in the coupling procedure after rehydration The stock protein concentration is possibly low Make sure the protein concentration is correct and the recommended amount is coupled to the microspheres The stock protein suspension possibly contains foreign proteins azide glycine Tris or some primary amines Dialyze the protein of interest to remove the competing substances The reaction volumes and or mixing method may be incorrect The final reaction volume for the 2 hr incubation is critical for successful coupling Refer to the protocol End to end mixing during the 2 hr incubation is very important Please use an optical rotator The anti species detection antibody may not be correct Make sure the anti species detection antibody is correct for example if you are using a mouse monoclonal to couple to the microspheres use an anti mouse detection antibody Make sure the anti species detection antibody has the appropriate label on it either biotin or phycoerythrin Page 18 of 20 Page 19 of 20 rower IMAP INSIDE TECHNOLOGY EZZEZA Case
5. 00 12000 Lo WJU MMRRETNTa RM M 10000 8000 6000 4000 Median Fluorescence 2000 Anti Species IgG PE conjugate ug ml Page 16 of 20 Troubleshooting This troubleshooting guide may be helpful in solving problems that may arise For more information call Technical Support toll free at 1 877 785 BEAD 2323 Outside of U S and Canada call 1 512 381 4397 Send e mail inquiries to support luminexcorp com stick to the inner surfaces of some tubes Problem Possible Cause s Recommendations Low Bead Uncoupled Make sure to use tubes supplied in this Count microspheres tend to kit only Microspheres were lost during washes Use the recommended magnetic separator to perform all washes See Recommended Material list Use the transfer pipette to remove all supernatant during washes The microsphere pellets are loose and using a pipettor could disturb the bead pellet Incorrect probe height adjustment Adjust probe height according to the Luminex 200 FLEXMAP 3D or MAGPIX user manual Incorrect protocol set up on the Luminex instrument Make sure correct microsphere regions are selected based on your particular custom designed assay Page 17 of 20 Problem Possible Cause s Recommendations Low No The reagents may not Store all reagents as recommended signals have been stored correctly EDC may not have EDC must remain
6. 1 2 minutes and with the tube still positioned in the magnetic separator remove the supernatant with a transfer pipette 19 Calculate volume of antibody to be used in the reaction e Typically a range of 2 5 yg of antibody per 1x108 microspheres is a good starting point if performing the coupling reaction for the first time Example Calculation If coupling 5 million microspheres with an antibody at a concentration of 5 mg mL and using the suggested starting point of 5 ug of antibody per 1x1 08 microspheres of beads to couple Desired Ab conc Stock Ab conc Volume of Ab needed 5x 10 beads ES 1x10 beads 5 9 Volume of Ab needed Volume of Ab needed 5uL Page 11 of 20 20 Calculate the volume of Activation Buffer needed for the reaction e If coupling more than 5x108 microspheres subtract the volume of antibody calculated in Step 19 from 1000 uL e If coupling 5x10 microspheres or less subtract the volume of antibody calculated in Step 19 from 500 pL Example Calculation If 5 uL of the stock antibody is needed as calculated in the example in Step 19 for the coupling of 5 million microspheres Volume of Activation Buffer needed 1000uL or 500uL Volume of Ab needed Volume of Activation Buffer needed 500pL 5yL Volume of Activation Buffer needed 495uL 21 Add the appropriate volume of Activation Buffer calculated in Step 20 to the reaction tube 22 Add the appropri
7. Luminex xMAP Antibody Coupling Kit User Manual Product Catalog Number xMAP Antibody Coupling Kit 40 50016 For Research Use Only Not For Use In Diagnostic Procedures 89 00002 00 319 Rev B April 2011 Luminex Corporation 2001 2011 All rights reserved No part of this publication may be reproduced transmitted transcribed or translated into any language or computer language in any form or by any means without prior express written consent of LUMINEX CORPORATION 12212 Technology Boulevard Austin Texas 78727 6115 U S A Voice 512 219 8020 Fax 512 219 5195 xMAP Antibody Coupling Kit User Manual PN 89 00002 00 319 Rev B April 2011 Luminex Corporation Luminex reserves the right to modify its products and services at any time This guide is subject to change without notice Although prepared to ensure accuracy Luminex assumes no liability for errors or omissions or for any damages resulting from the application or use of this information This guide may be updated periodically To ensure that you have a current version access http www luminexcorp com support The most recent version of this guide is available for download at that URL The following are registered trademarks of Luminex Luminex Luminex 100 Luminex 100 IS Luminex 200 Luminex SD Luminex XYP FLEXMAP 3D xMAP xTAG Microspheres MagPlex MicroPlex MAGPIX and xPONENT
8. aging until needed and discarded after one use Reconstituted EDC should never be stored and reused All components are guaranteed up to the expiration date found on the label when stored in their original packaging and as specified in this manual The stability of coupled microspheres is dependent upon several factors including the protein stability and composition aseptic processing conditions presence of preservatives storage buffer storage temperature conditions etc However stability studies have shown that antibody coupled microspheres stored in appropriate storage buffer are stable over a period of 18 months Necessary Materials and Equipment There are various materials and pieces of equipment that are required for microsphere coupling which are not included with this kit Required Equipment e Luminex xMAP instrument MAGPIX LX100 200 or FLEXMAP 3D Luminex xPONENT software e Luminex MagPlex Microspheres Cat MC1XXXX 01 04 amp MC10XXX ID NOTE Visit http www luminexcorp com Products ReagentsMicrospheres MAG PLEX MICROSPHERES for a complete list of MagPlex microspheres and guide lines for selecting the right regions for your instrument e Luminex Instrument Performance Calibration and Verification Kits e Luminex Instrument Sheath Fluid LX100 200 amp FLEXMAP 3D or Drive Fluid MAGPIX Magnetic Separator Other Necessary Equipment Tube Rotisserie or Rotator e Water bath sonicator Page 6 of 20
9. ate volume of antibody calculated in Step 19 to the reaction tube 23 Vortex the reaction tube for a minimum of 10 seconds 24 Protect microspheres from light and rotate on rotator for 2 hours Rotation speed should be 15 30 rpm 25 Wash the microspheres A Place the reaction tube with microspheres into the magnetic separator for 1 2 minutes B With the tube still positioned in the magnetic separator remove the supernatant with a transfer pipette C Add 500 uL of Wash Buffer into the reaction tube D Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to disperse the microspheres 26 Repeat Step 25 twice for a total of three washes 27 Place the reaction tube with microspheres into the magnetic separator for 1 2 minutes and with the tube still positioned in the magnetic separator remove the supernatant with a transfer pipette 28 Add 1 mL of Wash Buffer into the reaction tube NOTE The Wash Buffer is used as a storage buffer after completing the coupling reaction 29 Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to disperse the microspheres 30 Protect from light and store at 2 8 C until needed Page 12 of 20 NOTE For optimal performance allow the coupled microspheres to block over night before first use Page 13 of 20 Coupling Assessment Once the coupling reaction has been completed the coupled microspheres can be enumerated and the efficiency of
10. ce opened the EDC solution must be pre pared and used quickly If performing multiple reactions be sure to prepare all of them prior to this step so that the EDC solution can be quickly added to all of the reaction tubes immediately after dissolution The EDC solution must be made fresh for each coupling event and the excess should be discarded Add EDC Solution to reaction tube e If coupling more than 5x108 microspheres add 50 uL of the EDC solution into the reaction tube e lf coupling 5x10f microspheres or less add 10 uL of the EDC solution into the reaction tube Vortex the reaction tube for a minimum of 10 seconds Protect microspheres from light and incubate at room temperature for 20 1 minutes and vortex the microspheres again for 10 seconds halfway through the incubation period Alternatively this incubation step can be done on a rotator at 15 30 rpm for 20 minutes without vortexing Page 10 of 20 16 Wash the microspheres A Place the reaction tube with microspheres into the magnetic separator for 1 2 minutes B With the tube still positioned in the magnetic separator remove the supernatant with a transfer pipette C Add 500 uL of Activation Buffer into the reaction tube D Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to disperse the microspheres 17 Repeat Step 16 twice for a total of three washes 18 Place the reaction tube with microspheres into the magnetic separator for
11. ed microsphere stocks to a final concentration of 50 beads yL in PBS 1 BSA NOTE Up to 4 different microsphere sets may be prepared in the same mixture in multiplex provided each set is a different microsphere region and the various antibodies coupled to those regions can be detected with the same detection anti body NOTE At least 1mL of the microsphere solution is required for each reaction NOTE Either PBS 1 BSA or PBS BN PBS 1 BSA 0 05 Azide pH 7 4 may be used as Assay Buffer Page 14 of 20 4 Prepare a solution of phycoerythrin labeled anti species IgG detection antibody at 4 g mL in PBS 1 BSA Prepare a 1 2 dilution series of that detection antibody solution to a concentration of 0 0625 g mL as shown in the following table Dilution Tube ue a A Antoan Detection Concentration 1 1 4 ug mL 1 2 500 uL 500 uL from Tube 1 1 2 g mL 1 4 500 uL 500 uL from Tube 1 2 1 pg mL 1 8 500 uL 500 uL from Tube 1 4 0 5 pg mL 1 16 500 uL 500 uL from Tube 1 8 0 25 pg mL 1 32 500 uL 500 uL from Tube 1 16 0 125 yg mL 1 64 500 uL 500 uL from Tube 1 32 0 0625 pg mL NOTE For optimal results use Costar round bottom 96 well plates and Luminex Magnetic Plate Separator with MagPlex microspheres 5 Aliquot 50 uL of the microsphere solution prepared in Step 3 into two entire columns of wells of the plate duplicate sets of 8 wells 16 wells total 6 Add 50 uL o
12. es at 15 30 rpm Page 8 of 20 4 Dispense the desired amount of microspheres from the stock vial into one of the microcentrifuge tubes reaction tube provided with the kit Example Calculation If coupling 5 million microspheres with microsphere stock concentration of 12 5x108 microspheres of beads to couple Volume of stock needed W ___ conc of stock vial 510 beads Volume of stock needed ____ _ 12 5x10 beads _ Volume of stock needed 0 4mL or 400 uL IMPORTANT This kit is designed for a maximum of 12 5x108 microspheres per reaction tube 5 Wash the microspheres A Place the reaction tube with microspheres into the magnetic separator for 1 2 minutes NOTE If performing multiple reactions simultaneously a microcentrifuge may be used in place of a magnetic separator to pellet the microspheres during wash steps Beads can be pelleted by microcentrifugation at gt 8000 x g for 1 2 minutes B With the tube still positioned in the magnetic separator remove the supernatant with transfer pipette NOTE For your convenience twenty disposable transfer pipettes have been included for the removal of supernatant during the various wash steps in this pro tocol If using one pipette for the removal of Activation Buffer and one for the removal of Wash Buffer the kit should have enough to perform up to ten individual reactions i e 2 rxn Special care should be taken t
13. f PBS 1 BSA as a blank sample into the wells in Row A containing the microsphere solution 7 Add 50 uL of each of the diluted detection antibody solutions prepared in Step 4 into the appropriate wells of the plate as shown in the plate layout below 1 2 3 4 5 6 7 8 9 10 11 12 A Blank Blank B 164 1 64 c 132 1 32 D 116 1 16 E 18 18 F 14 14 G 12 1 2 H 11 1 1 Example plate layout using columns 1 amp 2 Page 15 of 20 Mix the reactions gently by pipetting up and down several times with a pipettor Cover the plate and incubate for 30 minutes at room temperature on a plate shaker Clip the plate in place on the Luminex Magnetic Plate Separator and rapidly and forcefully invert over a biohazard receptacle to evacuate the liquid from the wells NOTE For information on the MagPlex Manual Wash Method please visit http www luminexcorp com Products ReagentsMicrospheres MAGPLEX MICROSPHERES Wash each well with 100 uL of PBS 1 BSA by gently pipetting up and down several times with a pipettor and remove the liquid by using the procedure described in the previous step Repeat the previous wash step once Resuspend the microspheres in 100 uL of PBS 1 BSA by gently pipetting up and down several times with a pipettor Analyze 50 75 uL on the Luminex analyzer according to the system manual An example of typical results is shown below ANTIBODY COUPLING CONFIRMATION 140
14. l groups on the surface of the microspheres to form an active O acylisourea intermediate This intermediate forms a more stable ester using Sulfo NHS N hydroxysulfosuccinimide The ester reacts with the primary amines NH2 groups of antibodies to form a covalent bond amide linkage In the protocol described in this manual the Sulfo NHS is added to the reaction prior to the addition of EDC to maximize efficiency of the reaction The presence of Sulfo NHS in the mixture at the time of the EDC addition is critical due to the limited stability of the EDC microsphere conjugate The reaction with the carboxylated microsphere does not begin until EDC is added to the mixture Page 4 of 20 Protein Considerations This kit includes a specially formulated Activation Buffer pH 6 0 which is suitable for most antibodies The protein to be coupled must be free of sodium azide bovine serum albumin BSA glycine tris hydroxymethyl aminomethane Tris glycerol or amine containing additives and should be suspended in phosphate buffered saline PBS pH 7 4 A number of buffers can be used successfully in this coupling reaction Generally the pH at which a coupling reaction occurs should be compatible with the solubility of the protein of interest This should be considered when coupling different proteins Additionally coupling efficiency will vary depending on a variety of factors including type of antibody quantity of antibody quantity of micro
15. o avoid cross contamination when performing multiple reactions simultaneously C Add 500 uL of Activation Buffer into the reaction tube D Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to dis perse the microspheres 6 Repeat Step 5 once for a total of two washes Page 9 of 20 10 14 15 Place the reaction tube with microspheres into the magnetic separator for 1 2 minutes and with the tube still positioned in the magnetic separator remove the supernatant with a transfer pipette Add Activation Buffer into the reaction tube e If coupling more than 5x108 microspheres add 400 yL of Activation Buffer into the reaction tube e If coupling 5x108 microspheres or less add 480 uL of Activation Buffer into the reaction tube Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to disperse the microspheres Vortex the provided Sulfo NHS tube for a minimum of 10 seconds Add Sulfo NHS solution to the reaction tube e If coupling more than 5x108 microspheres add 50 uL of the Sulfo NHS solution into the reaction tube lf coupling 5x108 microspheres or less add 10 uL of the Sulfo NHS solution into the reaction tube Add 250 pL of Activation Buffer into the 10 mg vial of EDC Invert the EDC vial and then vortex the vial for 10 12 seconds to dissolve the EDC IMPORTANT EDC will begin to degrade once exposed to moisture in the atmo sphere and the Activation Buffer On
16. ory tract and skin irritation Introduction The xMAP Antibody Coupling AbC Kit contains all of the reagents necessary to covalently couple antibodies to Luminex MagPlex microspheres beads in approximately three hours This kit can also be used to couple other proteins to Luminex microspheres but due to the large diversity in protein composition coupling performance with other proteins is not guaranteed For more information on protein coupling please visit http Awww luminexcorp com support Coupling is achieved through carbodiimide reactions involving the primary amino groups on the antibody or protein of choice and the carboxyl functional groups on the microsphere surface This kit is configured for a one time use and contains enough reagent to couple as few as 2 5x108 or as many as 50x108 microspheres The kit can be used to perform as many as en individual coupling reactions at scales of 2 5x1 06to 5x108 microspheres per reaction or as many as four reactions at scales of up to 12 5x1 0 microspheres per reaction Scale Number of beads rxn Reactions 5x109 or less 10 Up to 12 5x10 4 IMPORTANT Luminex strongly recommends that the EDC reagent be discarded after one use Each coupling reaction requires a minimum of 2 5x108 microspheres The coupled microspheres can then be used with a Luminex xMAP instrument to develop monoplex or multiplex assays This kit is ideal for use in two modes
17. spheres etc As such it is recommended that several quantities of antibody input be tested to optimize the coupling reaction and functionality in the final assay If coupling for the first time 2 5j g of antibody per 1x1 0 microspheres is generally a good starting input The amount of antibody to use in the coupling reaction will depend on the quantity of coupled antibody necessary to promote optimal binding of the desired target molecule Kit Contents The following items are included in this kit Component Part Number Volume Mass Quantity EDC Reagent 11 40144 10 mg 1 ThermoSci 77149 Sulfo NHS 11 25169 250 uL 1 Activation Buffer 11 25171 45 mL 1 green cap Wash Buffer 11 25167 30 mL 1 1 5 mL tubes 11 00277 n a 10 Disposable pipettes 11 00321 nla 20 NOTE The EDC reagent is a Thermo Scientific product and is manufactured for Luminex for use in this kit For questions regarding the use of this product with this kit please contact Luminex technical support Page 5 of 20 Storage and Stability Photosensitive microspheres should be protected from light at all times All kit components are to be stored at 2 8 C The EDC reagent in its original packaging may be stored at 20 C to ensure the longest shelf life possible All other components should never be frozen IMPORTANT Due to the instability of the EDC reagent in solution it should always be stored in its original sealed pack
18. the coupling reaction assessed Microsphere Enumeration Although the protocol described in this manual will typically yield over a 90 recovery it is recommended that the user count the number of microspheres actually recovered after each coupling reaction This can be done with the use of a cell counter or hemacytometer Please refer to the cell counter or hemacytometer s users manual for appropriate instructions for doing so Coupling Confirmation It is strongly recommended to assess coupling efficiency before proceeding to assay development The coupled microspheres can be reacted with a phycoerythrin PE labeled target or antibody that binds to the coupled protein Alternatively the target or antibody may be biotinylated then labeled with PE This complex can then be analyzed on a Luminex xMAP instrument The intensity of the fluorescent signal of this reaction is directly proportional to the amount of protein on the surface of the microspheres This process provides a rapid assessment of the relative amount of protein coupled to the microspheres however this does not necessarily verify the functionality of the protein The ultimate test is the functional assay of the coupled protein Sample Coupling Confirmation Protocol Select the appropriate antibody coupled microsphere set or sets Resuspend the microspheres by vortex and sonication for approximately 20 seconds 3 Prepare a working microsphere solution by diluting the coupl
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