Luminex xMAP Antibody Coupling Kit - Manual (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
6
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 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 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.