Peptide microarrays on coated silicon slides for highly sensitive antibody detection.
Peptides, with their well-established chemistry and fully automated synthesis, provide an invaluable tool for the screening of protein ligands, for epitope mapping, and for antibody diagnostics on the microarray format.The method described in this chapter shows that the sensitivity of a peptide-based microimmunoassay is greatly improved by using a new, specifically developed substrate made of silicon coated by an optimized layer of silicon oxide. A set of six peptides corresponding to the sequences of human and rat acetylcholine receptor subunits was immobilized on glass and silicon slides coated by a copolymer of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl) propyl methacrylate, copoly(DMA-NAS-MAPS). The spotted probes were incubated with rabbit anti-sera and with purified antibodies raised against the corresponding peptides. The coated silicon slides, in comparison against the glass substrates, showed a five- to tenfold enhancement of the fluorescence signals, leading to the specific detection of the full set of antibodies down to a concentration of 0.5-1 ng/mL in serum. The sensitivity provided by the test allows its use for the diagnosis of antibodies in clinical samples.
Cretich
Marina
M
Istituto di Chimica del Riconoscimento Molecolare C.N.R, Milano, Italy.
Damin
Francesco
F
Longhi
Renato
R
Gotti
Cecilia
C
Galati
Clelia
C
Renna
Lucio
L
Chiari
Marcella
M
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology; D011108:Polymers / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry; D012825:Silicon / Q000737:chemistry*
D000906:Antibodies; D007106:Immune Sera; D010446:Peptide Fragments; D010455:Peptides; D011108:Polymers; D011978:Receptors, Nicotinic; D012825:Silicon