Peptide microarrays for serum antibody diagnostics.
Peptide microarrays are useful instruments for miniaturized high-throughput, high-content immunoassays. The substitution of linear epitopes of the protein antigen with short, synthetic peptides is virtually a straightforward approach to capture antigen-specific antibodies from serum samples; however, both the biologically active surface display of peptides and the establishment of a solidly performing peptide microarray immunoassay are often troublesome in detail. The following protocols aim to provide facilitated access to the production of a robust peptide microarray platform and an optimized analytical processing of peptide microarrays in serological diagnosis. The functional surface display is accomplished by site-specific immobilization of peptide probes in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde-coated substrate for microstructured surface immobilization of the probe complexes. The serological assay is based on the specific capture of primary antibodies in a sandwich complex between the surface-immobilized peptides and a fluorescently labelled secondary antibody. A proposal for the diagnostic evaluation of fluorescence data obtained with the peptide microarray is also made.
Andresen
Heiko
H
Fraunhofer Institut für Biomedizinische Technik, Institutsteil Potsdam, Potsdam, Germany.
Bier
Frank F
FF
D000906:Antibodies / Q000097:blood*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D001774:Blood Chemical Analysis / Q000295:instrumentation* / Q000379:methods; D018604:Epitope Mapping / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D040081:Protein Array Analysis / Q000295:instrumentation*; D012698:Serologic Tests / Q000295:instrumentation* / Q000379:methods
D000906:Antibodies