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


CONCLUSIONS
Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in speed, sample size, and quantification over standard ELISA-based titer methods.
van der Heyde
Henri C
HC
La Jolla Bioengineering Institute, La Jolla, California 92037, USA. hvande@san.rr.com
Burns
James M
JM
Weidanz
William P
WP
Horn
John
J
Gramaglia
Irene
I
Nolan
John P
JP
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood* / Q000276:immunology; D000953:Antigens, Protozoan / Q000097:blood / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D007132:Immunoglobulin Isotypes / Q000097:blood / Q000276:immunology; D008288:Malaria / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000097:blood / Q000276:immunology; D020066:Merozoite Surface Protein 1 / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D023421:Models, Animal; D008954:Models, Biological; D016788:Plasmodium chabaudi / Q000276:immunology; D015800:Protozoan Proteins / Q000097:blood / Q000276:immunology
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007132:Immunoglobulin Isotypes; D008565:Membrane Proteins; D020066:Merozoite Surface Protein 1; D015800:Protozoan Proteins; C090077:apical membrane antigen I, Plasmodium