Luminex xMAP Antibody Coupling Kit Manuel d'utilisateur télécharger pdf (Page 8)

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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

mide 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 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.

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Luminex xMAP Antibody Coupling Kit Manuel d'utilisateur Page 8