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 C December 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.
Safety.......................................................................................... 1 Introduction ................................................................................. 3 Method........................................................................................ 4 Protein Considerations................................................................... 5 Kit Contents ................................................................................ Storage and Stability...........
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.
Material Safety Data Sheets (MSDS's) are available upon request. Visit http://www.luminexcorp.com/support for more information. 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 respiratory 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://www.luminexcorp.
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 fortyfive 96-well plates (@ 2,500 beads/well) for every 12.5x106 microspheres coupled. Method 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.
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.
Storage and Stability Photosensitive microspheres should be protected from light at all times. All kit components are to be stored at 2-8ºC. 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 packaging until needed and discarded after one use.
• Vortexer • Pipettor Optional Equipment • xMAP Antibody Coupling Kit Quick Guide (89-30000-00-361) (Available at http://www.luminexcorp.
Instructions General Workflow Coupling Protocol The following is the detailed step-by-step process for coupling antibodies (or similar proteins) to carboxylated magnetic microspheres. An abridged quick reference guide (part number 89-30000-00-361) can be found online at http://www.luminexcorp.com/ downloads. IMPORTANT: Protect photosensitive microspheres from light whenever possible throughout this entire procedure. 1. 2. 3.
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 12.5x106 microspheres: IMPORTANT: This kit is designed for a maximum of 12.5x106 microspheres per reaction tube. 5. Wash the microspheres. A. Place the reaction tube with microspheres into the magnetic separator for 1-2 minutes.
7. 8. 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. • If coupling more than 5x106 microspheres, add 400 μL of Activation Buffer into the reaction tube. • If coupling 5x106 microspheres or less, add 480 μL of Activation Buffer into the reaction tube. 9.
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 μL 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.
20. Calculate the volume of Activation Buffer needed for the reaction. • If coupling more than 5x106 microspheres, subtract the volume of antibody calculated in Step 19 from 1000 μL. • If coupling 5x106 microspheres or less, subtract the volume of antibody calculated in Step 19 from 500 μL.
NOTE: For optimal performance allow the coupled microspheres to block overnight before first use.
Coupling Assessment Once the coupling reaction has been completed, the coupled microspheres can be enumerated and the efficiency of 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.
Prepare a solution of phycoerythrin-labeled anti-species IgG detection antibody at 4 μg/mL in PBS-1% BSA. Prepare a 1:2 dilution series of that detection antibody solution to a concentration of 0.0625 μg/mL as shown in the following table. 4. Volume of PBS-1% BSA Dilution Tube Volume of Detection Antibody Concentration 1:1 - - 4 μg/mL 1:2 500 μL 500 μL from Tube 1:1 2 μg/mL 1:4 500 μL 500 μL from Tube 1:2 1 μg/mL 1:8 500 μL 500 μL from Tube 1:4 0.
8. Mix the reactions gently by pipetting up and down several times with a pipettor. 9. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker. 10. 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/MAGPLEXMICROSPHERES. 11.
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. Problem Possible Cause(s) Recommendations Low Bead Count Uncoupled microspheres tend to stick to the inner surfaces of some tubes Make sure to use tubes supplied in this kit only.
Problem Possible Cause(s) Recommendations Low/No signals The reagents may not have been stored correctly Store all reagents as recommended. EDC may not have been stored correctly or rehydrated long before use EDC must remain in the pouch it is 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.
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