Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics.

Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.

A complete substitutional analysis of VIP for better tumor imaging properties.

Since numerous tumor cells overexpress the vasoactive intestinal peptide (VIP) receptor subtype 1 (VPAC(1)), VIP-dye conjugates would be useful as contrast agents for in vivo imaging. However, proteolytic degradation of VIP in vivo limits their diagnostic use and highlights the need for structurally optimized VIP derivatives with improved pharmacokinetics. Here, we applied parallel nano-synthesis of cleavable peptides on cellulose membranes to perform a complete VIP substitutional analysis. The resulting 504 different VIP-dye analogs were tested for cell binding by flow cytometry. They provided a detailed analysis of amino acid positions essential for binding to VPAC(1) overexpressing cells. A generalized VIP-dye binding motif derived from the substitutional analysis results served as a reference point for further optimization. An [Arg8]-VIP-dye analog showed increased stability towards proteolytic degradation, good tumor-to-tissue contrast in mice and a longer half-life in vivo.