A generic surface chemistry for peptide microarrays implementation: application to the detection of anti-H3 antibody.
Peptide microarray can be implemented by immobilization of peptides on a solid support or by direct on-chip peptide synthesis (OCPS). In the first case, peptide primary sequences can be ensured prior to their immobilization but structural diversity is achieved at high cost in terms of reagents. In the second case, high diversity is achieved with low amount of reagents but the primary and secondary structures cannot be ascertained. In both cases, the immobilization step will influence the overall biological activity. We proposed a strategy where direct peptide on-chip synthesis and peptide immobilization are viewed as complementary approaches. In a first step, OCPS is envisioned for the screening and selection of biologically relevant peptides. In a second step, selected peptides would be synthesized on resin, qualified and immobilized for implementing microarrays (i.e. for diagnosis). A versatile surface chemistry for both OCPS and peptide immobilization was developed allowing for an identical physico-chemical environment for both implementation strategies. In the present report, a 16 mer peptide corresponding to the human histone H3 epitope was synthesized on an amino-functionalized support. Surface stability (including upon deprotection steps) and peptide primary and secondary structures were assessed with Cy3-streptavidine conjugates and with immunoassays. Peptides, either on-chip synthesized or immobilized, exhibited a similar biological activity.
El Khoury
Graziella
G
Université de Lyon, Institut des Nanotechnologies de Lyon, UMR CNRS 5270, Site Ecole Centrale de Lyon, 36 Avenue Guy de Collongue, 69134 Ecully, Cedex, France.
Laurenceau
Emmanuelle
E
Chevolot
Yann
Y
Souteyrand
Eliane
E
Cloarec
Jean-Pierre
JP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011817:Rabbits; D013499:Surface Properties
D000906:Antibodies; D000939:Epitopes; D005456:Fluorescent Dyes; D006657:Histones; D010446:Peptide Fragments