Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.

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Cashew nuts are an increasingly common cause of food allergy. We compare the soluble protein profile of cashew nuts following heating. SDS-PAGE indicate that heating can alter the solubility of cashew nut proteins. The 11S legumin, Ana o 2, dominates the soluble protein content in ready to eat and mildly heated cashew nuts. However, we found that in dark-roasted cashew nuts, the soluble protein profile shifts and the 2S albumin Ana o 3 composes up to 40% of the soluble protein. Analysis of trypsin-treated extracts by LC/MS/MS indicate changes in the relative number and intensity of peptides. The relative cumulative intensity of the 5 most commonly observed Ana o 1 and 2 peptides are altered by heating, while those of the 5 most commonly observed Ana o 3 peptides remaine relatively constant. ELISA experiments indicate that there is a decrease in rabbit IgG and human serum IgE binding to soluble cashew proteins following heating. Our findings indicate that heating can alter the solubility of cashew allergens, resulting in altered IgE binding. Our results support the use of both Ana o 2 and Ana o 3 as potential cashew allergen diagnostic targets.

A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal-to-noise ratios.

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