Molecularly imprinted polymer as a synthetic antibody for the biorecognition of hazelnut Cor a 14-allergen

Artificial receptors that mimic their natural biological counterparts have several advantages, such as lower production costs and increased shelf-life stability/versatility, while overcoming the ethical issues related to raising antibodies in animals. In this work, the proposed tailor-made molecularly imprinted polymer (MIP)-allergen receptors aimed at substituting or even transcending the performance of biological antibodies. For this purpose, a MIP was proposed as an artificial antibody for the recognition of hazelnut Cor a 14-allergen. The target protein was grafted onto the conducting polypyrrole receptor film using gold screen-printed electrodes (Au-SPE). The electrochemical assessment presented a linear response for the dynamic range of 100 fg mL-1-1 μg mL-1 and a LOD of 24.5 fg mL-1, as determined by square wave voltammetry from the calibration curves prepared with standards diluted in phosphate buffer.
Surface plasmon resonance (SPR) was used as a secondary transducer to evaluate the performance of the Cor a 14-MIP sensor, enabling a linear dynamic range of 100 fg mL-1– 0.1 μg mL-1 and a LOD of 18.1 fg mL-1. The selectivity of the tailored-made Cor a 14-MIP was tested against potentially cross-reactive joplink GAPDH Monoclonal Antibody plant/animal species based on the rebinding affinity (Freundlich isotherm-KF) of homologues/similar proteins, being further compared with custom-made polyclonal anti-Cor a 14 IgG immunosensor.
Results evidenced that the MIP mimics the biorecognition of biological antibodies, presenting higher selectivity (only minor cross-reactivity towards walnut and Brazil nut 2S albumins) than the Cor a 14/anti-Cor a 14 IgG immunosensor. The application of electrochemical Cor a 14-MIP sensor to model mixtures of hazelnut in pasta enabled quantifying hazelnut down to 1 mg kg-1 (corresponding to 0.16 mg kg-1 of hazelnut protein in the matrix). To the best of our knowledge, Cor a 14-MIP is the first sensor based on an artificial/synthetic biorecognition platform for the specific detection of hazelnut allergens, while presenting high-performance parameters with demonstrated application in food safety management.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus

Background and aim: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein.
Materials and methods: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a).
Results: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies.
Conclusion: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

Detection of Aspergillus flavus in Wheat Grains Using Anti-mannoprotein (MP1) and Spore Protein Polyclonal Antibodies

Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies.
The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross-reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains, these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

Enzymatic Hydrolysis and Fermentation of Pea Protein Isolate and Its Effects on Antigenic Proteins, Functional Properties, and Sensory Profile

Combinations of enzymatic hydrolysis using different proteolytic enzymes (papain, Esperase, trypsin) and lactic fermentation with Lactobacillus plantarum were used to alter potential pea allergens, the functional properties and sensory profile of pea protein isolate (PPI). The order in which the treatments were performed had a major impact on the changes in the properties of the pea protein isolate; the highest changes were seen with the combination of fermentation followed by enzymatic hydrolysis. SDS-PAGE, gel filtration, and ELISA results showed changes in the protein molecular weight and a reduced immunogenicity of treated samples.
Treated samples showed significantly increased protein solubility at pH 4.5 (31.19-66.55%) and at pH 7.0 (47.37-74.95%), compared to the untreated PPI (6.98% and 40.26%, respectively). The foaming capacity was significantly increased (1190-2575%) compared to the untreated PPI (840%).
The treated PPI showed reduced pea characteristic off-flavors, where only the treatment with Esperase significantly increased the bitterness. The results from this study suggest that the combination of enzymatic hydrolysis and lactic fermentation is a promising method to be used in the food industry to produce pea protein ingredients with higher functionality and a highly neutral taste. A reduced detection signal of polyclonal rabbit anti-pea-antibodies against the processed protein preparations in ELISA furthermore might indicate a decreased immunological reaction after consumption.

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