Luminex xMAP Antibody Coupling User Manual

xMAP® Antibody

Product Catalog Number

xMAP

Antibody Coupling Kit

Not For Use In Diagnostic Procedures

Coupling Kit User Manual

40-50016

For Research Use Only

89-00002-00-319

Rev C

December 2011

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

Antibody Coupling Kit User Manual

xMAP

PN 89-00002-00-319 Rev. C

December 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 IS™, Luminex® 200™, Luminex® SD™, Luminex® XYP™, FLEXMAP

3D™, xMAP xPONENT

, xTAG® Microspheres, MagPlex®, MicroPlex®, MAGPIX™, and

, Luminex® 100™,

Safety.......................................................................................... 1

Introduction ................................................................................. 3

Method........................................................................................ 4

Protein Considerations................................................................... 5

Kit Contents ................................................................................ 5

Storage and Stability................................................................... 6

Necessary Materials and Equipment.......................................... 6

Instructions ................................................................................. 8

General Workflow........................................................................... 8

Coupling Protocol........................................................................... 8

Coupling Assessment ............................................................... 14

Microsphere Enumeration............................................................ 14

Coupling Confirmation.................................................................. 14

Sample Coupling Confirmation Protocol...................................... 14

Troubleshooting........................................................................ 17

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

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.

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.

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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.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

or as many as 50x106 microspheres. The kit can be used to perform as many as

2.5x10 ten individual coupling reactions at scales of 2.5x10

; or as many as four reactions at scales of up to 12.5x106 microspheres per

reaction reaction.

and contains enough reagent to couple as few as

to 5x106 microspheres per

Scale

(beads/rxn)

5x106 or less

Up to 12.5x10

IMPORTANT:

Number of Reactions

* Luminex strongly recommends that the EDC reagent be discarded after one use. ** Each coupling reaction requires a minimum of 2.5x106 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:

• 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.

• 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.

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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 12.5x10

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.

The coupling procedure involves a two-step carbodii on the surface of the polystyrene microspheres must first be activated with a carbodiimide derivative prior to coupling the antibody. EDC (1-Ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride) reacts with the carboxyl 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.

mide reaction. The carboxyl groups

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