The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with FT-ICR MS and the results compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:monoclonal antibody (Ag-mAb) complexes. MAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen-antibody complex in solution.

Exosomes are nanosized (30–150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 μm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen–antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

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