African swine fever (ASF) is an acute, highly contagious and deadly infectious disease. It is a threat to animal health with major potential economic and societal impact. Despite decades of ASF vaccine research, still some gaps in knowledge are hindering the development of a functional vaccine. Worth mentioning are gaps in understanding the mechanism of ASF infection and immunity, as well as the fact that – in case of this disease – virus proteins, so-called protective antigens, responsible for inducing protective immune responses in pigs are not identified yet. In this paper we elaborate on a methodology to identify protective antigens based on epitope mapping by microarray technology. High density peptide microarrays, combined with fluorescence scanning, have been used to analyze the interaction of peptide sequences of African swine fever virus (ASFV) proteins with antibodies present in inactivated serum from infected and healthy animals. The study evidenced ASFV proteins already under the radar for vaccine development, such as p54, and identified specific sequences in those proteins that may become the focus for future vaccine candidates. Such methodology is amenable to automation and high-throughput and may help developing better targeting for next generation vaccines.
Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.
Multifunctional CMOS Microchip Coatings for Protein and Peptide Arrays
Stadler, Volker; Beyer, Mario; König, Kai; Nesterov, Alexander; Torralba, Gloria; Lindenstruth, Volker; Hausmann, Michael; Bischoff, F. Ralf; Breitling, Frank
Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules. The coated microchips were compatible with the harshest conditions emerging in microarray generating methods, thoroughly retaining structural integrity and microelectronic functionality. Nonspecific adsorption of proteins on the chip’s surface was completely obviated even with complex serum protein mixtures. We could demonstrate the background-free antibody staining of immobilized probe molecules without using any blocking agents, encouraging further integration of CMOS technology in proteome research.
