Phage Displayed Domain Antibodies (dAb) for Detection of Allergenic Pistachio Proteins in Foods

Pistachio nuts (Pistacia vera) have been consumed by past and present-day civilizations because of their organoleptic characteristics and potential health benefits. However, they can also produce moderate to severe IgE-mediated reactions in allergic individuals. In this work, we report the isolation of the first recombinant antibodies against pistachio nut, produced without animal immunization, to be used in immunoassays for detection of allergenic pistachio in food products. Several phage display biopanning strategies were evaluated to screen the human-based domain antibody library (dAb) in search for pistachio-specific probes. The clone producing the PVF4 phage-dAb was finally selected, and it does not cross-react with cashew despite the phylogenetic proximity with pistachio. Western blot and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOF) analysis demonstrated that this clone recognised a unique band of ∼22 kDa related to the basic subunit of pistachio 11S globulin (allergen Pis v 2). The PVF4 phage-dAb allowed detection of pistachio in a food matrix with a limit of detection (LOD) of 3983 mg kg-1 in an indirect phage-enzyme-linked immunosorbent assay (ELISA). The ELISA method developed was used to assess applicability of the PVF4 phage-dAb for analysis of 77 commercial food products.

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis.

The primary structure and function of nucleoside diphosphate kinase (NDK), a substrate non-specific enzyme involved in the maintenance of nucleotide pools is also implicated to play pivotal roles in many other cellular processes. NDK is conserved from bacteria to human and forms a homotetramer or hexamer to exhibit its biological activity. However, the nature of the functional oligomeric form of the enzyme differs among different organisms. The functional form of NDKs from many bacterial systems, including that of the human pathogen, Mycobacterium tuberculosis (MtuNDK), is a hexamer, although some bacterial NDKs are tetrameric in nature. The present study addresses the oligomeric property of MsmNDK and how a dimer, the basic subunit of a functional hexamer, is stabilized by hydrogen bonds and hydrophobic interactions. Homology modeling was generated using the three-dimensional structure of MtuNDK as a template; the residues interacting at the monomer-monomer interface of MsmNDK were mapped. Using recombinant enzymes of wild type, catalytically inactive mutant, and monomer-monomer interactive mutants of MsmNDK, the stability of the dimer was verified under heat, SDS, low pH, and methanol. The predicted residues (Gln17, Ser24 and Glu27) were engaged in dimer formation, however the mutated proteins retained the ATPase and GTPase activity even after introducing single (MsmNDK- Q17A, MsmNDK-E27A, and MsmNDK-E27Q) and double (MsmNDK-E27A/Q17A) mutation. However, the monomer-monomer interaction could be abolished using methanol, indicating the stabilization of the monomer-monomer interaction by hydrophobic interaction.