RESUMEN
There are a number of examples of immunologic cross-reactivity elicited by pollens, fruits, seeds, and nuts of closely related plant species. Such cross-reactivity is of particular concern for patients with food allergies. In this report, we investigated a spice (mahleb) that is prepared from the kernel of the St. Lucie cherry, Prunus mahaleb, for cross-reactivity with almond (Prunus dulcis), using enzyme-linked immunosorbent assay (ELISA) and Western blot. Almond and mahleb are members of the same genus. Cross-reactivity between the mahleb and almond was demonstrated by reaction of cherry and almond kernel protein extracts with antibodies raised against almond proteins. Almond-specific murine monoclonal IgG, rabbit polyclonal IgG, and almond-allergic serum IgE each exhibited cross-reactivity with cherry kernel protein. Because of the demonstrated cross-reactivity between almond and mahleb, these findings should be of special concern to almond-allergic patients and attending medical personnel.
Asunto(s)
Hipersensibilidad a los Alimentos/inmunología , Inmunoglobulina G/análisis , Nueces/inmunología , Prunus avium/química , Prunus dulcis/inmunología , Especias/análisis , Alérgenos/análisis , Alérgenos/inmunología , Animales , Western Blotting , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/inmunología , Ratones , Nueces/química , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Prunus avium/inmunología , Prunus dulcis/química , Conejos , Semillas/química , Semillas/inmunologíaRESUMEN
The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/metabolismo , Biología Molecular/métodos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Animales , Biotina/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Proteínas de Escherichia coli/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Células 3T3 NIH , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genéticaRESUMEN
The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments.
Asunto(s)
Alérgenos/química , Anacardium/química , Medición de Intercambio de Deuterio/métodos , Mapeo Epitopo/métodos , Epítopos/química , Proteínas de Almacenamiento de Semillas/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Epítopos/inmunología , Espectrometría de Masas/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Almacenamiento de Semillas/inmunologíaRESUMEN
The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.