Analysis of antigen-specific antibodies and their isotypes in experimental malaria.

ABSTRACT

BACKGROUND: Measuring antibody production in response to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi -quantitative analysis of antibody production in response to malaria infection. METHODS: We used microspheres as solid supports on which to capture and analyze circulating antibodies. Antigen immobilized on beads captured antigen-specific antibodies for semi- quantitative analysis using fluorescent secondary antibodies. Anti-immunoglobulin antibodies on beads captured specific antibody isotypes for affinity estimation using fluorescent antigen. RESULTS: Antigen-mediated capture of plasma antibodies enables determination of antigen-specific antibody "titer," a semi-quantitative parameter describing a convolution of antibody abundance and avidity, as well as parameters describing numbers of antibodies bound/bead at saturation and the plasma concentration-dependent approach to saturation. Results were identical in single-plex and multiplex assays, and in qualitative agreement with similar parameters derived from ELISA-based assays. Isotype-specific antibody-mediated capture of plasma antibodies allowed the estimation of the affinity of antibody for antigen. CONCLUSION: Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in speed, sample size, and quantification over standard ELISA-based titer methods.