Large-scale profiling and monitoring of antibody repertoires is possible through next generation sequencing (NGS), phage display libraries and microarrays. These methods can be combined in a pipeline, which ultimately maps the antibody reactivities onto defined arrays of structures – peptides or carbohydrates. The arrays can help analyze the individual specificities or can be used as complex patterns. In any case, the targets recognized should formally be considered mimotopes unless they are proven to be epitopes driving the antibody synthesis. Here, the advantages and disadvantages of the major profiling techniques as well as their current and future application in disease prediction and vaccination are discussed.
Purification and Characterization of Anacardium occidentale (Cashew) Allergens Ana o 1, Ana o 2, and Ana o 3
Reitsma, Marit; Bastiaan-Net, Shanna; Sforza, Stefano; van der Valk, Johanna P. M.; van Gerth van Wijk, Roy; Savelkoul, Huub F. J.; de Jong, Nicolette W.; Wichers, Harry J.
In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.
Combinatorial Synthesis of Peptide Arrays onto a Microchip
Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
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.
