Amide-containing neoepitopes: the key factor in the preparation of hapten-specific antibodies and a strategy to overcome.

For a long time, people have suffered from uncertainty, complexity, and a low success rate in generating and screening antibodies against small molecules, which have become the core bottlenecks of immunochemistry. Here, the influence of antigen preparation on antibody generation was investigated at both molecular and submolecular levels. Neoepitopes (amide-containing neoepitopes) formed in the preparation of complete antigens are one of the most important factors limiting the efficiency of generating hapten-specific antibodies, which was verified by different haptens, carrier proteins, and conjugation conditions. Amide-containing neoepitopes present electron-dense structural components on the surface of prepared complete antigens and, therefore, induce the generation of the corresponding antibody with much higher efficiency than target hapten. Crosslinkers should be carefully selected and not overdosed. According to these results, some misconceptions in the conventional anti-hapten antibody production were clarified and corrected. By controlling the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) during the synthesis of immunogen to limit the formation of amide-containing neoepitopes, the efficiency of hapten-specific antibody generation could be significantly improved, which verified the correctness of the conclusion and provided an efficient strategy for antibody preparation. The result of the work is of scientific significance in the preparation of high-quality antibodies against small molecules.

Fabrication of copolymer brushes grafted superporous agarose gels: Towards the ultimate ideal particles for efficient affinity chromatography.

A composite immobilized-metal affinity agarose particle was designed for the selective separation and purification of histidine-tagged proteins from complicated biological samples. The composite particle was constructed using superporous agarose particles as supporting matrix, flexible copolymer brushes as scaffolds to render higher ligand densities, and Ni2+-chelated iminodiacetic acids as recognition elements. Superporous agarose composite particles endow high permeability and interfering substance tolerance. The copolymer brush was prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and glycidyl methacrylate, followed by iminodiacetic acids and Ni2+ ions. The physical and chemical properities of the composite particle were thoroughly investigated. The composite particles were shown to be able to selectively separate histidine-tagged recombinant proteins in the presence of high quantities of interfering chemicals in a model protein-binding experiment. By altering the temperature, the protein binding of the composite particles can be modulated. The superporous agarose particles supported polymer brush enables fast and efficient separation and purification of target proteins with high permeability, low backpressure, and high interfering matrix tolerance, which pave the path for bioseparation through designing and fabrication of novel agarose particles-based functional materials.

Investigation of COVID-19 Impact on the Food and Beverages Industry: China and India Perspective.

The sudden breakout of coronavirus disease (COVID-19) rapidly spread across the globe, leaving no country behind in being affected by the global pandemic in the year 2019-20. As COVID-19 commenced, within months two major Asian giants initiated the norms of social distancing and lockdowns in their societies. The indiscriminate nature of the current pandemic has not only impacted the health and quality of life of people but has also disrupted the global economy, supply chains, and countries all over the world. In food and beverage manufacturing industries, the unanticipated disruption has encumbered its lock on the global food supply chain and service sector as major cities shut down for several months in China and India. Human existence is dependent upon food, which renders energy for activity, growth, and all functions of the body. Although both China and India have shown eminent response to tackle the ongoing pandemic, the food supply chain remains vastly exposed to significant COVID-19 risks. This research primarily investigates the ongoing COVID-19 scenario in two major economies (China and India), delivering insight into the pandemic's impact within the food and beverage manufacturing sectors, and explores the policies adopted and strategies undertaken for sustainability in food supply chains.

Immunostimulatory and allergenic properties of emulsified and non-emulsified digestion products of parvalbumin (Scophthalmus maximus) in RBL-2H3 cells and BALB/c mouse models.

In the present study, the influence of lipid emulsion on the allergenicity of digestion products of fish parvalbumin (PV) was investigated, which was initially subjected to simulated gastric/intestinal digestion both under emulsified and non-emulsified conditions. The release of ß-hexosaminidase (ß-hex), histamine (His), tryptase (TPS), interleukin 4 (IL-4), and IL-13 in RBL cells was decreased by 79.32, 26.19, 41.67, 53.95 and 54.40%, respectively, following stimulation with the gastric digestion products of PV. Whereas, lipid emulsified digestion products of PV (e-PV) significantly enhanced the release of active mediators and cytokines. The digestion products of emulsified PV at 180 min resulted in a higher release of ß-hex (197.60%), His (12.18%), TPS (38.85%), IL-4 (48.19%) and IL-13 (59.40%), as compared to that of PV. However, no obvious differences in the release of active substances and cytokines were noted between intestinal digestion products of PV and intestinal digestion products of emulsified PV. In the mouse model studies, digested PV products reduced the anaphylactic scores, whereas e-PV manifested a higher level of allergic symptoms. Moreover, mice treated with 50% e-PV had significantly higher levels of specific IgE (32.56%), total IgE (16.67%) and total IgG1 (5.15%) than those treated with 50% PV. Mice treated with 50% e-PV had significantly higher levels of His (8.50%) and TPS (10.07%) compared with mice treated with 50% PV. Lipid emulsions altered the digestibility of PV in gastrointestinal digestion and enhanced the allergenicity of PV digestion products at the cellular levels, subsequently posing a higher risk of allergic reactions in susceptible individuals.

Comparison of digestibility and potential allergenicity of raw shrimp (Litopenaeus vannamei) extracts in static and dynamic digestion systems.

In this work, a simplified dynamic digestion system was developed, and used for comparing the digestibility and potential allergenicity of raw shrimp extracts (RSE) in static and dynamic digestion systems. Protein hydrolysis was analyzed by electrophoresis, and the potential allergenicity was reflected in IgG/IgE binding ability and activation of basophils. In comparison with static digestion, protein hydrolysis indicated different kinetic behaviors, especially tropomyosin (TM) showed better digestion stability during dynamic digestion. The potential allergenicity of RSE exhibited different changing trends with digestion in the two systems. However, the apparent molecular weight (Mw) of immune fragments (>11 kDa) showed good approximation, and the IgE-binding fragment near 70 kDa revealed outstanding digestion stability than primordial protein in both systems. In conclusion, the dynamic conditions had a significant impact on the assessment of the persistence and potential allergenicity of digestion-resistant allergens, while the apparent Mw of IgG/IgE binding hydrolysate was not affected.

Tyrosinase/caffeic acid cross-linking alleviated shrimp (Metapenaeus ensis) tropomyosin-induced allergic responses by modulating the Th1/Th2 immunobalance.

In this study, the effect of enzymatic cross-linking of shrimp tropomyosin (TM) with tyrosinase and caffeic acid (TM-Tyr/CA) on the allergic response were assessed using in vitro and in vivo models. The RBL-2H3 and KU812 cell lines were employed to evaluate the changes in the stimulation abilities of TM-Tyr/CA that showed significant inhibition of mediators and cytokines. The digestibility of cross-linked TM was improved and the recognitions of IgG/IgE were markedly reduced, as revealed by western blotting. TM-Tyr/CA decreased anaphylactic symptoms, and hindered the levels of IgG1, IgE, histamine, tryptase and mouse mast-cell protease-1 (mMCP-1) in mice sera. Cross-linked TM downregulated the production of interleukin (IL)-4, IL-5, and IL-13 by 51.36, 12.24 and 20.55%, respectively, whereas, IL-10 and IFN-γ were upregulated by 20.71 and 19.0%. TM-Tyr/CA showed reduced allergenicity and may have preventive effect in relieving TM induced allergic response via immunosuppression and positive modulation of T-helper (Th)1/Th2 immunobalance.

Visual detection of tropomyosin, a major shrimp allergenic protein using gold nanoparticles (AuNPs)-assisted colorimetric aptasensor.

A gold nanoparticle-based label-free colorimetric assay was developed to detect the shrimp allergenic protein tropomyosin (TM), an important biomarker responsible for severe clinical reactivity to shellfish. In a gold nanoparticles (AuNPs)-tropomyosin-binding aptamer (TMBA) complex, the aptamer adsorbs onto the surface of AuNPs and dissociates in the presence of TM. In addition, AuNPs tend to aggregate in the presence of ionic salt, revealing a color change (i.e., wine-red to purple/blue) with a shift in the maximum absorption peak from 520 nm. In the presence of specific binding TM, the aptamer folds into a tertiary structure where it more efficiently stabilizes AuNPs toward the salt-induced aggregation with a hypsochromic shift in the absorption spectra compared to the stabilized AuNPs by aptamer alone. Based on the aggregation and sensitive spectral transformation principle, the AuNPs-based colorimetric aptasensor was successfully applied to detect TM with a range of 10-200 nmol/L and a low detection limit of 40 nmol/L in water samples. The reliability, selectivity, and sensitivity of the aptasensor was then tested with food samples spiked with TM. The observed detection limit was as low as 70 nmol/L in shrimp, 90 nmol/L in tofu, and 80 nmol/L in eggs, respectively. We anticipate the proposed AuNPs-based colorimetric aptasensor assay possesses a high potential for the easy and efficient visual colorimetric detection of TM. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-020-00085-5.

Hapten-Branched Polyethylenimine as a New Antigen Affinity Ligand to Purify Antibodies with High Efficiency and Specificity.

Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR-PEI) was conjugated to CNBr-Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR-PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL-18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.

An overview on marine anti-allergic active substances for alleviating food-induced allergy.

Food provides energy and various nutrients and is the most important substance for the survival of living beings. However, for allergic people, certain foods cause strong reactions, and sometimes even cause shock or death. Food allergy has been recognized by the World Health Organization (WHO) as a major global food safety issue which affect the quality of life of nearly 5% of adults and 8% of children, and the incidence continues to rise but there is no effective cure. Drug alleviation methods for food allergies often have shortcomings such as side effects, poor safety, and high cost. At present, domestic and foreign scientists have turned to research and develop various new, safe and efficient natural sources of hypoallergenic or anti-allergic drugs or foods. There are many kinds of anti-allergic substances obtained from the plants and animals have been reported. Besides, probiotics and bifidobacteria also have certain anti-allergic effects. Of all the sources of anti-allergic substances, the ocean is rich in effective active substances due to its remarkable biodiversity and extremely complex living environment, and plays a huge role in the field of anti-food allergy. In this paper, the anti-food allergic bioactive substances isolated from marine organisms encompassing marine microbial, plant, animal sources and their mechanism were reviewed and the possible targets of anti-allergic substances exerting effects are illustrated by drawing. In addition, the development prospects of marine anti-allergic market are discussed and forecasted, which can provide reference for future research on anti-allergic substances.

Boronic acid-functionalized agarose affinity chromatography for isolation of tropomyosin in fishes.

BACKGROUND: Tropomyosin is now receiving increasing attention because of its significant allergenic activity in various fishery products but its simple and effective isolation still remains a challenging task. RESULTS: An agarose-based boronate affinity chromatography was produced for the first time to isolate tropomyosin in various fishery products using 3,5-difluoro-4-formyl-phenylboronic acid as the functional monomer, tris(2-aminoethyl)amine as the multi-branched ligand, and agarose gel particles as supporting materials. The agarose concentration, binding pH, and the concentration of elution buffers demonstrated significant effects on separation performance. Under optimized conditions, the purity of the isolated tropomyosin was higher than 90%, with the column adsorption capacity over 1.85 mg mL-1 and the enrichment efficiency over 65%. Such efficiency was also validated with different fish samples including Paralichthys olivaceus, Thunnusthynnus, Oreochromis spp., and Lophius litulon. CONCLUSION: In comparison with conventional methods, the established affinity chromatography demonstrated excellent biocompatibility (without involving any organic solvent), better speed (from at least 1-2 days to 3-4 h), and simplicity (from at least five steps to three steps). This suggests that it is a novel and promising technique for the isolation of tropomyosin and other glycoproteins (including most allergens) in foodstuffs. © 2019 Society of Chemical Industry.

Preparation of a novel polyethyleneimine functionalized sepharose-boronate affinity material and its application in selective enrichment of food borne pathogenic bacteria.

Detection of pathogenic bacteria by polymerase chain reaction (PCR) is progressively emerging, although it is still hindered by a complex matrix, long-term bacterial enrichment and low bacterial abundance. Here, we report a novel material based on boronate affinity for recognition and enrichment of bacteria using a pre-PCR method. After optimization, the material exhibited high boronate affinity toward bacteria, with adsorption capacities of S. aureus and Salmonella spp. incubated in 0.01 M PBS (pH 7.4) at 37 °C for 15 min calculated as (906.60 ±â€¯15.73) × 107 cfu/g and (582.59 ±â€¯13.19) × 107 cfu/g, respectively, without any bacterial death during the binding process. The material was then applied to enrich S. aureus and Salmonella spp. from the spiked water and 25% cow milk samples followed by mPCR, which resulted in high bacterial enrichment and demonstrated great potential for selective enrichment of bacteria in food samples.

Development of a method for the quantification of fish major allergen parvalbumin in food matrix via liquid chromatography-tandem mass spectrometry with multiple reaction monitoring.

The availability of analytical methods for quantification of allergens is crucial for the correct assessment and labeling of products in order to protect allergic consumers. For this purpose, a simple, sensitive and accurate technique was developed based on liquid chromatography-tandem mass spectrometry and multiple reaction monitoring mass spectrometry (LC-MRM-MS/MS). The proposed method uses a simple purification with heat and a completely optimized tryptic digestion. This method has been validated according to the requirements defined by ICH (Q2 [R1]), having a linear range from 0.10 to 1179.36 nM with r > 0.999. The parvalbumin beta in flounder (Paralichthys olivaceus) has been quantified at a low level down to 0.10 µg/g with satisfactory precision (RSD < 18.35%) and accuracy (<13.3%). The new approach was successfully applied for the determination of parvalbumin beta in the other food matrices.

Effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on protein oxidation and textural properties of fish mince (Pagrosomus major) during frozen storage.

BACKGROUND: Frozen storage of minced fish is currently one of the most important techniques to maintain its functional properties. However, some deterioration does occur during frozen storage and cause quality loss. The effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on lipid and protein oxidation and textural properties of red sea bream (Pagrosomus major) during 90 days of frozen storage at -18 °C were investigated. RESULTS: All added antioxidants at 1 g kg-1 resulted in significantly lower thiobarbituric acid-reactive substances (TBARS) compared to the control during the 45 days of frozen storage. The antioxidants were also effective in retarding protein oxidation concerning to total sulfhydryl content and protein carbonyl content. Brown seaweed polyphenols and α-tocopherol significantly retarded the inactivation of Ca2+ -ATPase activity during the first 45 days, whereas ascorbic acid had no such effect. The antioxidant activity showed either an invariable or decrease trend after 45 days storage. Adding antioxidants had a significant effect on the breaking force of the gels during the frozen storage period. CONCLUSION: These results indicate that brown seaweed polyphenols and α-tocopherol can be used to prevent oxidative reactions and thus maintain the structure of the gel formed by fish mince during frozen storage. © 2016 Society of Chemical Industry.

Effect of malonaldehyde cross-linking on the ability of shrimp tropomyosin to elicit the release of inflammatory mediators and cytokines from activated RBL-2H3 cells.

BACKGROUND: Malonaldehyde, the primary by-product of lipid peroxidation in food, modifies the structural and functional properties of proteins by cross-linking. The aim of this study was to investigate the effect of malonaldehyde on the allergenicity of shrimp tropomyosin. RESULTS: RBL-2H3 cells, a model of type I allergic reactions, were sensitised with sera from patients allergic to shrimp, and were stimulated with native and cross-linked tropomyosin. Release of inflammatory mediators such as ß-hexosaminidase, histamine, tryptase, cysteinyl leukotriene, and prostaglandin D2 was clearly suppressed in a manner that depended on the extent of tropomyosin cross-linking. Release of interleukin-4 (IL-4) and IL-13 was similarly decreased. Notably, cells sensitised with one patient's serum released IL-4 at comparable levels in response to native and cross-linked tropomyosin. CONCLUSION: Cross-linking strongly modulates the ability of shrimp tropomyosin to induce release of inflammatory cytokines and mediators from activated RBL-2H3 cells. © 2016 Society of Chemical Industry.