Analysis of Antibody Reactivity to Malaria Antigens by Microsphere-Based Multiplex Immunoassay

Protein multiplex assays enable serological analysis of multiple target proteins simultaneously, using relatively small volumes of patient sample per assay. Here we present a detailed protocol to analyze antibody reactivity to malaria antigens by microsphere-based multiplex assay (xMAP technology). This method involves coupling of recombinant proteins to fluorescently labeled microspheres; simultaneous exposure of all microspheres to plasma or sera, and detection of antigen-specific antibodies with a fluorescent labeled anti-human Fc region antibody. In addition to total IgG, this assay can be adapted to measure multiple properties of the antibody Fc region, including subclass, isotype, and Fc receptor or complement C1q binding.