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In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute res

Data used for the analysis of pepsin interaction with polystyrene microplastic of 10 and 100 µm in size. Binding isotherms data were used for the construction of binding isotherms from which binding between pepsin and polystyrene microplastic was described. Structural features of pepsin in the presence of polystyrene were analysed by CD spectrometry and spectrofluorimetry.

Research data for the unpublished paper: Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties. Densitometry analysis of ovalbumin band intensity after digestion in the presence of microplastics on SDS-PAGE gels.

The experimental data for the results shown in the manuscript "Microplastic contamination of edible parts of commercially important mussels, clams and shrimps in different markets". Statistical investigation of the presence of microplastics in bivalve species using microFTIR. This dataset includes samples from various origins, including Sebia, South Korea, Croatia and Belgium, and includes species as diverse as shrimp, mussels and clams.

BACKGROUND: Phycocyanobilin (PCB) is an open-chain blue tetrapyrrole chromophore of C-phycocyanin (C-PC), a major chromoprotein derived from the cyanobacterium Arthrospira platensis having numerous health-promoting effects. Relying on the ability of PCB to attach to the sulfhydryl group of proteins, we propose a new method for covalent attachment of PCB to bovine serum albumin (BSA) as a means of its functionalization. RESULTS: Traut's reagent (TR, 2-iminothiolane), modifying lysine residues, was used to optimize the introduction of sulfhydryl groups in BSA. A higher degree of BSA thiolation by TR induces more profound alterations of its structure, resulting in minor oligomerization and aggregation. A 50-fold molar excess of TR was found to be the optimal, balancing thiolation level and adverse effect on protein structure. PCB was covalently attached to newly introduced sulfhydryl groups at pH 9 at 20-fold PCB/BSA ratio. An increase in the TR/BSA molar ratio leads to increased efficiency of PCB conjugation with thiolated BSA. Compared to native BSA, BSA–PCB conjugate binds quercetin with similar affinity but has higher antioxidant activity and increased oxidative stability. CONCLUSIONS: PCB-modified BSA could serve as a stable, food-compatible carrier of bioactive PCB, but also bind other ligands that would be protected from oxidative damage due to the high antioxidant potential of covalently bound PCB. Thiolation by TR is, at the same time, a simple method for the covalent functionalization of virtually any protein by bioactive PCB or for obtaining PCB-based fluorescent probes.

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno- PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

In the last several decades, the trend of seafood consumption has significantly increased not only in the countries with a tradition of seafood consumption, but also in other ones [1]. The increase in the world's population and the awareness of healthy food, the globalization of markets, and the development of aquaculture are some of the factors that have led to this trend. The aquaculture of shellfish like clams, mussels, oysters and scallops has been very developed all around the world and the food products based on them have become part of the daily diet for many consumers. In addition, these food products are considered healthy food because of the high content of proteins and essential fatty acids, but their consumption may carry some risks of food allergy. Tropomyosin from shellfish (TPM) is the major allergen responsible for the development of anaphylaxis in persons with food allergy. The content of TPM in shellfish and its bioavailability from food products can have potential influence on the sensitization of consumers to TPM. It is known that food processing can change the bioavailability of food allergens [2]. The main goal of this study was the investigation of how processing and packaging of shellfish samples can affect the content of TPM in them. For this study, clam Venerupis philippinarum was chosen as the species with the highest world aquaculture production [1]. After the purchasing of live clams, the animals were separated into 5 groups for the next treatments: fresh live (control group), freshly removed inner content was kept at +4°C for 3 days (three days` shelf-life), frozen in a plastic bag and kept at -20 °C during 7 days, marinated and kept in a glass jar at room temperature during 8 days and freshly boiled. After processing and packaging of samples, the total protein extracts were prepared in 10 mM sodium phosphate buffer pH 7.4 1M NaCl, 1 mM PMSF and the concentration of total proteins was determined by BCA method. The concentration of TPM i

Accurately diagnosing people with suspected SARS-CoV-2 infection is essential to help manage COVID-19. Currently available SARS-CoV-2 diagnostics detect either RNA of the virus by RT-PCR or the presence of viral antigens in biological fluids by ELISA or similar techniques. Low sensitivity of antigen tests could lead to the risk of false negative results. Therefore, this study aimed to develop a highly sensitive immuno-PCR method for quantifying SARS-CoV-2 nucleocapsid (N) protein that combines the specificity of sandwich ELISA with the sensitivity of PCR. Recombinant N protein fragment was produced in E. coli as an expression system and purified using immobilized metal ion affinity chromatography. The antibodies against the N protein were raised in rabbits and mice. High-affinity polyclonal mice and rabbit N protein-specific antisera were purified using ammonium sulfate precipitation and used to develop sandwich ELISA for the quantification of N protein. Mice polyclonal serum was used as a capture for N protein. N protein bound to mice antibodies was detected with rabbit polyclonal sera. A double-stranded amino-DNA molecule of 77 base pairs was PCR-synthesized, covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. The results were compared to analogous sandwich ELISA consisting of alkaline phosphatase-labeled goat anti-rabbit antibody. The sensitivity of immuno-PCR for quantification of N protein was increased by up to 7-fold compared to analogous ELISA, having a limit of detection of 92 pg/mL and a limit of quantification of 840 pg/mL. The developed immuno-PCR method thus has the potential to be used as a new antigen test for COVID-19 and beyond.

In the last 50 years, the annual per capita consumption of seafood products worldwide has more than doubled, from almost 10 kg in 1960 to over 20 kg in 2014. Seafood protein is an essential part of the diet in many countries, particularly where total protein intake is low [1]. However, as defined by the European Community, fish, and shellfish tropomyosins (TPM) are major allergens and major causes of anaphylaxis [2]. The increasing prevalence of food allergies is consistent with the increasing pollution of soil and water with plastic particles. To investigate the potential link between increasing plastic pollution and increasing food allergy prevalence, we aim to develop methods for precise and accurate monitoring of allergens and plastic in real seafood samples. TPM was isolated from shrimp (Litopenaeus vannamei), clams (Venerupis philippinarum), and mussels (Mytilus galloprovincialis). The obtained in-house TPM proteins from three different sources were resolved using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The concentration of TPM in seashell samples from different geographical origin was determined using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with prior optimization of adequate TPM standard curve using commercial and non-commercial in-house prepared TPM standards. TPM standards resolved via 2D-PAGE revealed the presence of two isoforms of shrimp and mussels TPM standard, one dominant and one less abundant isoform. Two isoforms from both seafood sources, shrimp and mussels, are slightly different in molecular weight and pI value. As for the TPM standard obtained from clams, the 2D electrophoregram showed possibly eight isoforms with small differences in mass and pI values. Furthermore, the presence of three dominant isoforms can be observed that differ slightly in molecular mass, while other isoforms also differ in pI value. The ELISA results, regarding TPM standard curve optimization, showed that in both the commercial shrimp

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno-PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating accuracy as low as 19.8 pg/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.

Microplastics is abundant in the environment, food and beverages and get ingested by humans. Its complex interplay with proteins lead to formation of corona. Tightly bound proteins represent hard corona, while weaker binding partners are found in soft corona. Separation of hard and soft corona of allergenic proteins of shrimps, eggs and cow’s milk, tropomyosin (TPM), ovalbumin (OVA) and beta-lactoglobulin (BLG) and identification of binding partners by proteomics was aim of our study. Allergenic proteins were purified from egg white, shrimps and cow’s milk. Binding to polyethylene terephthalate microplastics (PET) (70-100 m) was probed at pH 7 for purified allergens and egg white proteins. After establishment of binding equilibrium, soft and hard corona were separated and analyzed by SDS PAGE, followed by identification of bound proteins by nanoLC-HRMS. Binding of all allergenic proteins was observed in both soft and hard corona. Soft corona contains exclusively intact, full length OVA, TPM and BLG. Hard corona is enriched for truncated OVA and oligomers of TPM. OVA fragments are partially or fully enfolded and have higher level of exposed hydrophobic patches resulting in higher affinity for PET microplastics. In comparison to OVA and TPM, hard corona of BLG is less abundant under similar conditions. BLG is compact globular protein with lower level of exposed hydrophobic patches in comparison to ovalbumin and tropomyosin. In hard corona, trace amounts of contaminating alfa-lactalbumin become enriched. In the presence of egg white protein extract OVA forms both SC and HC on microplastics, being the dominant protein of hard corona (with ovotransferrin). Lysozyme and ovomucin are present only in hard corona. Both proteins are known for their strong bioactivity and represent a small fraction of total egg white proteins. Our results show that allergenic proteins form hard corona on PET microplastics. Among egg white proteins, minor proteins such as lysozyme and ovomucin

Novi korona virus (SARS CoV-2) koji se pojavio u Vuhanu 2019. godine pripada grupi jednolančanih RNK virusa [1]. Predstavlja novi infektivni agens za humanu populaciju i veoma je brzo detektovan u velikom broju zemalja. Uzročnik je respiratornih infekcija koje mogu da budu praćene i veoma teškom kliničkom slikom. Brzo širenje, odsustvo imuniteta na ovaj virus i odsustvo pouzdanih testova za detekciju virusa u trenutku izbijanja pandemije su bolest izazvanu ovim virusom brzo pretvorili u zdravstveni i društveni problem najvišeg prioriteta na globalnom nivou. Iako su najveće biotehnološke kompanije ubrzano počele sa razvojem i masovnom proizvodnjom dijagnostičkih testova i vakcina, njihova dostupnost u trenucima najveće potražnje je i dalje nedovoljna, a cene istih su limitirajući faktor za bolju kontrolu bolesti i širenja pandemije [2]. Razvoj sopstvenih i održiva proizvodnja testova i vakcina za COVID-19 su od velikog društvenog značaja. Važan preduslov za održivu proizvodnju testova je dostupnost rekombinantnih antigena virusa i mogućnost proizvodnje istih na velikoj skali za potrebe proizvodnje domaćih testova. Ovim tehničkim rešenjem se opisuje dobijanje dva ključna antigena novog korona virusa rekombinantnom tehnologijom i njihova primena u serološkom ELISA testu koji proizvodi Institut za primenu nuklearne energije, INEP, kao i za dobijanje reagenasa za detekciju antigena novog korona virusa (specifičnih antitela). U prvoj fazi, optimizovane su sekvence proteina koje su podigle osetljivost postojećih seroloških testova. Inovativnost našeg pristupa se ogleda i u razrađenim eksperimentalnim protokolima za dobijanje rekombinantnih proteina nukleokapsida na velikoj skali, kao i u solubilnoj formi, što olakšava postupak prečišćavanja. Izbor fragmenta nukleokapsida koji se heterologo eksprimira u solubilnoj formi, a specifično detektuje antitela i generiše jak imuni odgovor tokom imunizacije životinja (imunogenost) na osnovu pregleda poznatih epitopskih sekvenci je k

Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno- PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating a ccuracy a s l ow a s 1 9.8 p g/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.

rom over 10 kg in 1960 to over 20 kg in 2014, the yearly per capita consumption of marine products has increased significantly during the past 50 years. In many nations, especially those with poor overall protein intake, seafood protein is a crucial component of the diet1. However, as defined by the European Community shellfish protein tropomyosin (TPM) is one of the major allergens and major causes of anaphylaxis 2. Although TPM originating from vertebrates is not considered as an allergen there is evidence that several fish tropomyosin can be allergenic3. TPM protein is organized of two parallel alpha-helical molecules which are wound around each other forming a coiled -coil structure2. Although the degree of similarity between TPM molecules is high, their allergenic potency is different. Scientists putting a lot of effort into iden tifying and sequencing tropomyosin isoforms since this information probably explains the intriguing nature of the TPM molecule. This is a very challenging task given that the differences in mass and pI values between TPM isoforms are discrete. TPM was isolated from mussels (Mytilus galloprovincialis), and clams (Venerupis philippinarum) according to the protocol developed within/and for purposes of the IMPTOX research project. The obtained “in-house” TPM proteins were resolved using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Isoelectric focusing was performed on rehydrated Immobiline strips IPG 3-5.6NL 13 cm in Ettan IPGPhor 3 device. The second dimension was carried out on 12% PAA gels. Upon protein bands excision to prevent TPM interchain disulfide cross-linking reduction and alkylation of cysteine was performed. The samples were digested with proteomics-grade trypsin in a 1:50 enzyme-to- substrate ratio overnight at 37 °C. The obtained peptides were chromatographically separated using the EASY-nLC II system and analyzed using Orbitrap Exploris 240 mass spectrometer. 2D-PAGE resolved two isoforms of mussel TPM and

Seafood has been widely recognized as an integral part of a balanced and healthy diet, thanks to its high nutritional value and associated multiple health benefits. Seafood contamination with microplastics (MPs) have recently been highlighted as an emerging concern for global food security.1 According to definition, polymer particles with a diameter of less than 5 mm are referred to as microplastics particles (MPs)2. Most bivalves, as well as mussels, are filter feeders and can accumulate particles in their bodies. By consuming seafood, MP particles can be included in the human food chain. Most of the studies analyzed MPs content in environmental samples, instead of point-of-sales samples. The aim of this study was to determine the abundance of microplastics in mussels collected from Croatia seafood market intended for human consumption. 5 clean laboratory criteria (100% cotton lab coat, clean glassware, filtered solutions, rinsed the outside of animals before dissection, and limited air exposure of samples) were fulfilled by our study. Digestion and manipulation of the sample was conducted in a clean hood. Procedural controls were included for each measurement. After extensive removal of biological material by alkaline, enzymatic and oxidation digestion, MPs were characterized by micro-Fourier-transform infrared spectroscopy (Micro-FTIR). It is a proven method for the identification of all types of polymers and allows a reliable differentiation between substances of natural and synthetic origin. Our approach included one individual per sample for micro-FTIR analysis, no sub-grouping of the sample for chemical characterization (100% of particles isolated from each sample have been analyzed by micro- FTIR) and manual confirmation of all recorded spectra with more than 60% match to the polymer material in the spectral library (Thermo-Fisher; using the OMNIC PictaTM software). Soft tissue from inside the shell was not extensively washed to allow estimation of MPs intak

Experimental data for the results shown in the manuscript Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR are given in the attachment.

Size distributions (by intensity percentage) of NPs washed suspended in different dispersants, water,BSA (0.05%),SDS (0.1 %). Distributions data from different combined fractions: from NP-10 to NP-30; from NP-40 to NP-60 and from NP-70 to NP-90. Output from the Malvern zetasizer Nano-ZS ZEN 3600 (Malvern Panalytical, Malvern, UK).

The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. 1H NMR spectra of the NPs preparation before and during all the washing steps. Determination of SDS Level in Corona of PET NPs by 1H NMR. Output from the Varian/Agilent NMR 400 MHz. NMR spectra were processed in Mnova software.

Determination of hydrodynamic diameter of PET NPs washed and dispersed in water, BSA (0.05%), and SDS (0.1%), and NPs unwashed and dispersed in water. Zeta potential, mobility, and conductivity determination in PET NPs at different stages of purification. Output from the Malvern zetasizer Nano-ZS ZEN 3600 (Malvern Panalytical, Malvern, UK).

The analysis of structural changes of pepsin in the presence of polystyrene microplastic of 10 and 100 µm in size in simulated gastric fluid. Data obtained from J-815 CD spectropolarimeter, and further analyzed by Origin and Excel software.

Percentage of hemolysis of RBC in the presence of washed PET NPs and unwashed NPs (with SDS) determined by incubation of human red blood cells (RBCs) from three healthy donors. Output from the Shimadzu UV/ViS 1800 (Kyoto, Japan) nspectrometer - raw spectral data and the calculation of percentages of hemolysis (procesesd data).

Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the difference

Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the difference

Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja za suzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostima ELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studije je bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih seruma za rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjam apsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testa proizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima i ze evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumi visokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog za ovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adherirani na dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli ite koncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu za kvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jim poliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe uje spektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikaciju N-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototip ELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ 10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekciju N-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela. Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u za kvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma., The accurate diagnosis of people with suspected infection with the SARS-CoV-2 is essential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can be detected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens in biological fluids in ELISA or similar techniques using antibodies developed in animals. The aim of the study was the establishment of a quantitative polyclonal sera-based test for routine measurement of the concentration of SARS CoV-2 nucleocapsid protein using absorbance measurement in a standard 96-well microtiter plate. For the purposes of the test development, recombinant N protein was produced and used for the production of mice and rabbit antisera. Produced antisera were purified and titer was determined. High-affinity polyclonal N-protein specific antisera were used for N-protein specific ELISA test development. The test is based on mice polyclonal sera adhered to microtiter plate bottom for the capture of the N protein from the specimen. Various concentrations of the recombinant N-protein were used to generate a standard curve for protein quantification. The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera and anti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement. We have successfully developed the prototype ELISA for the quantification of N-protein with the detection limit being in the range of ng/mL. The average LOD value for the prototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was 10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for the detection of N-protein with affinity and specificity similar to, or better than commercial antibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence for quantification of the N-protein in protein-rich samples, similar to human sera.

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Tropomyosin (TPM) is considered as a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products iscrucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDSPAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content., Tropomiozin (TPM) se smatra glavnim alergenom morskih plodova. Razvoj metoda za kvantifikaciju TPM-a u prehrambenim proizvodima je ključan za alergične osobe. Nekoliko pufera za ekstrakciju je upoređeno u pogledu efikasnosti ekstrakcije protein iz sveže zamrznutih i kuvanih gambora, tokom 2 i 24 časa. Sadržaj proteina u solubilnim ekstraktima je određen Bredfordovom metodom. Proteinski profil ekstrakata je analiziran SDS-PAGE metodom. Za kvantifikaciju TPM-a je razvijena sendvič ELISA metoda. Nijedan puffer nije pokazao značajnu razliku u količini ekstrahovanih protein nakon 2 i 24 časa ekstrakcije. U poređenju sa sirovim, značajno manje proteina je ekstrahovano iz kuvanih gambora. Kvantifikacija TPM-a ELISA metodom je pokazala da se fosfatom puferisanim fiziološkim rastvorom (PBS) koji sadrži 1 M NaCl (PBSN) i karbonatnim puferom, pH 10, ekstrahuje oko 6 puta više TPM-a u poređenju sa PBS-om. Ovo ukazuje na značaj odabira adekvatnog pufera za ekstrakciju TPM-a, jer se upotrebom tradicionalno korišćenih pufera može potceniti koncentracija TPM-a u gamborima.

Tropomyosin (TPM) is considered a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products is crucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDS-PAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content.