Multiplexed quantification of human IgG, IgA, and IgM with the FlowMetrix system.

Abstract

A quantitative assay for the three major serum immunoglobulins (Igs) has been developed for the FlowMetrixTM system that performs each measurement simultaneously in the same test tube. The FlowMetrix system is a computer-enhanced flow cytometer that utilizes multiple fluorescent color sets of microspheres to provide a multiplexed format (1)(2). The microspheres are 5.5 μm in diameter and are composed of polystyrene and methacrylate to provide a surface carboxylate functionality. They are dyed internally with a mixture of two fluorophores that enables their classification into 64 distinct color sets. Igs are found in serum at high concentrations, allowing their assay in the competitive inhibition format. Each assay was developed by the attachment of purified IgG, IgA, or IgM to three of the available 64 uniquely fluorescing microsphere sets. Antibody specifically reactive to each Ig class was then labeled with a third fluorophore that acted as the reporter molecule. The amount of this reporter detected on the microsphere surface was inversely proportional to the concentration of soluble Ig in the serum sample. Each assay was conveniently optimized by altering the concentration of reporter antibody to provide a dynamic range encompassing the …