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Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species
Full text linkIn 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.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_859
Detekcija alergena u hrani metodama molekularne biologije - Susam (Sesamum indicum L.) – Kvalitativna detekcija specifičnih DNK sekvenci tehnikom real time PCR
No full textNovo tehničko rešenje primenjeno na međunarodnom nivou M81;
U rešenju MNO za biotehnologiju i poljoprivredu u kojem se tehničko rešenje svrstava u kategoriju M81 došlo je do greške u kucanju, pa je tako jedan od autora tehničkog rešenja Luka Dragačević, a ne Luka Dragičevi
Combination of arrythmogenic right ventricular cardiomyopathy with loeys-dietz syndrome: case report
No full textArrhythmogenic right ventricular dysplasia is a hereditary cardiomyopathy - a common cause of sudden cardiac death in children and young adults. Loeys-Dietz syndrome is an ultra-rare connective tissue disorder characterized by aneurysms of the aorta and other large arteries, arterial tortuosity, and joint hypermobility and is associated with pathogenic variants in genes encoding protein components TGF-β pathway. We present a rare case of a two-abovementioned genetic disorders combination in a proband with a complex and rapidly progressive cardiovascular syndrome.Аритмогенная кардиомиопатия правого желудочка - наследственная кардиомиопатия, частая причина
внезапной сердечной смерти у детей и молодых взрослых. Синдром Лоиса-Дитца - редкое наследственное
заболевание соединительной ткани, характеризующееся аневризмой аорты и других крупных артерий,
патологической извитостью артерий и гипермобильностью суставов, связанное с патогенными вариантами
в генах, кодирующих белковые компоненты TGF-β сигнального пути. Представляем случай сочетания двух
вышеупомянутых наследственных заболеваний у пробанда с комплексным и быстро прогрессирующим
поражением сердечно-сосудистой системы
Supplementary information for the article: Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.
Full text linkS1.1. 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 respiratory syndrome coronavirus 2, 46 kDa 504.9 74.22 183 mass of purified N protein suggesting that the obtained sera is highly specific for N protein (Figure 2). Section S3 Diagnostic validationS3.1. Stabilization of capture antibodies Pre-coated ELISA plates were prepared for usage in clinical practice. To ensure the preservation of the biofunctionality of the surface-bound capture antibodies, the commonly used stabilizing excipient, 3% sucrose with 10% glycerol in MilliQ water was used. The plates were incubated with 300 μL per well of a stabilizing agent for 1 hour at room temperature. After an hour of incubation, the solution was carefully aspirated from each well. The plate was then blotted against clear paper towels to remove any remaining liquid, and the plates were allowed to air dry for 3 hours at RT. Dried plates were wrapped in parafilm and stored at 4 °C for later use. To remove the stabilizing agent coating, wells were washed with slightly acidic distilled water (pH of 6) three times, leaving the plate prepared for subsequent assay steps. Section S4. Characterization of N protein by HRMS S4.1. SDS PAGE and in-gel digestion Characterization of the produced recombinant N protein was done by HRMS after its in-gel digestion. A total of 10 μg of purified protein(s) were loaded in a 0.5 cm wide well and after SDSPAGE gel was stained with Coomassie Brilliant Blue R-250 (CBB). Protein gel bands were washed, reduced with dithiothreitol, and alkylated with iodoacetamide, followed by in-gel trypsin digestion1 (Shevchenko et al. 2006) with some minor modifications. The amount of trypsin was leveled to a trypsin/sample ratio of 1:30 (w/w). The final concentration of MS-grade trypsin (diluted in 25 mM ammonium bicarbonate buffer) was 1 ng/μL. Sample clean-up was performed using zip tips HyperSep C18 (Thermo Fisher Scientific Inc., Bremen, Germany). S5.1 Immunization of rabbits and mice Mice immunization Swiss Webster mice (n=10) were immunized subcutaneously with N protein formulated with Complete Freund`s adjuvant (CFA; 1st dose, 100 μg N protein / dose) or Incomplete Freund`s adjuvant (IFA; 2nd and 3rd doses, 50 μg N protein / dose) in three-week intervals. Mice were housed in small groups of up to six animals and had access to commercial mice food and water ad libitum. N protein solution (500ug/ml in PBS) was sterilized by filtering through 0.22 um filters. Sterile N protein solution was mixed with CFA (Sigma, Cat. No. F5881) at ratio 1:1 (v/v) under aseptic conditions. In total 400 ul of N protein-CFA emulsion (N protein final concentration 250ug/ml) was applied per immunization per mouse. Initial immunization was done by injection of N protein in CFA given subcutaneously (SC) in four sites (thigh pocket, base of tail, and mediastinum) with a 100 ul using 23-25 gauge needle. In total 100 ug of N protein was applied per mouse (25 ug per site). Subsequent immunizations with booster doses were done in the same way, but using IFA (Sigma, Cat. No. F5506) instead of CFA and N protein final concentration was 125 ug/ml. . In total 50 ug of N protein was applied per mouse (12.5 ug per site). Immunizations were done every three weeks. Mice immunization scheme: 1. day 0 – N protein in PBS: CFA = 1:1 (v/v); N protein final concentration was 250 μg/mL; 400 μL per mice (4x100 μL), e.g. 100 μg per mice 2. day 21 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μL per mice (4x100 μL), e.g. 50 μg per mice 3. day 42 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μl per mice (4x100 μL), e.g. 50 μg per mice First bleeding was performed two weeks after the 3rd dose, and then in intervals not shorter than two weeks. The sera obtained after the first bleeding was tested for the production of specific anti-N protein antibodies.Related to the published version: [https://intor.torlakinstitut.com/handle/123456789/858]Supplementary material for: [https://doi.org/10.3390/ijms25010333
Inactivation of diphtheria toxin by site-directed mutagenesis
No full textDiphtheria toxin is a single polypeptide chain
produced by toxigenic strains of Corynebacterium diphtheriae that causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis.
Formaldehyde (chemical) detoxification converts diphtheria toxin into toxoid, which is used
in diphtheria vaccine production. Recombinant,
genetically detoxified diphtheria toxin is superior in terms of safety and purity, but it has still
not found its application in recombinant diphtheria vaccine production. Both chemically and
genetically inactivated forms of the diphtheria
toxin have proven effective as protein carriers in
conjugate vaccines. The goal of this study was to
create a plasmid construct which can be used to
express a genetically inactivated diphtheria toxin. Gene coding for diphtheria toxin was cloned
into pMALHisEk expression vector and introduced into DH5α competent Escherichia coli cells.
Three site-directed point mutations, which led to
three amino acid substitutions (G52E-substitutes
glycine with glutamic acid, G79D- substitutes
glycine with aspartic acid, E148D- substitutes
glutamic acid with aspartic acid) were conducted. A single G52E amino acid substitution is responsible for the loss of the enzymatic activity
of the diphtheria toxin. G79D is recognized as a
good candidate site for combining with other
mutations in vaccine development and E148D
may be a good candidate as carrier protein because it could reduce both the stability of NAD
binding and catalytic activity of the enzyme.
Each individual mutation is sufficient for toxin inactivation, but together they ensure non-toxicity,
preventing reversion to the wild-type sequence.
All mutations were confirmed by DNA sequencing. Recombinant diphtheria toxoid could serve
as a potential vaccine epitope or protein carrier
for conjugate vaccines. Further optimization of
recombinant protein expression in Escherichia
coli should provide sufficient quantities of soluble recombinant protein for further testing of its
safety, immunogenicity and protection
IgA monoclonal gammopathies are accompanied by higher total TGF-β1 levels than IgG or IgM monoclonal gammopathies
No full textThe progression of monoclonal gammopathies is affected by a range of factors, including the microenvironment surrounding plasma cells. It is recognized that TGF-β1 plays a distinct role in stimulating IgA production. Hence, this study aims to investigate whether individuals with serum IgA monoclonal immunoglobulins (paraproteins) exhibit elevated total TGF-β1 levels compared to those with IgG or IgM paraproteins. To achieve this goal, individuals with a positive laboratory finding of monoclonal gammopathy were segregated according to the paraprotein class as well as according to the type of the light chain. Total TGF-β1 levels were assessed in blood serum samples containing IgG (n = 50), IgA (n = 46), and IgM (n = 31) paraproteins. Elevated level of TGF-β1 was confirmed in sera with IgA paraproteins (median 25.8 ng/mL; interquartile range IQR: 19.0–33.7) compared to those having IgG (median: 18.2 ng/mL; IQR: 14.3–22.1; p < 0.001) or IgM paraproteins (21.5 ng/mL; IQR: 15.0–27.4; p = 0.043). Also, a higher TGF-β1 level was detected in sera with IgMλ than those with IgMκ paraproteins (p = 0.043). This research affirms the role of TGF-β1 in the pathophysiology of IgA monoclonal gammopathies and the potential switch towards the IgA isotype, known for a less favourable prognosis
Secrerome analysis of B. pertussis 8/84 vaccinal strain in different liquid media
No full textBordetella pertussis is an important human
pathogen causing serious infections in infants.
Acellular pertussis vaccines are composed of
purified secreted/surface proteins including inactivated pertussis toxin, as the most important
component. This study presents a secretome
analysis of one of the four strains included in
whole cell pertussis vaccine produced by Institute Torlak. For fermentation, three different
media were used; Cohen-Wheeler broth, casein
hydrolysate/yeast extract (CH), and casein acid
hydrolysate/yeast extract (CAH) based broths.
Upon cultivation for 48h, the bacteria were pelleted by centrifugation, prior to 60% ammonium
sulfate precipitation. The pellet was dissolved in
distilled water and subjected to methanol-chloroform precipitation. Proteomic investigation
of the precipitates were done by in-gel trypsin
digestion followed by label-free relative quantification on a nano LC-ESI-MS/MS system. Bacterial growth was similar in different growth media, but lower protein levels were detected in
Cohen-Wheeler broth. The mass spectrometric
analysis of CAH identified 143 proteins. Proteins
with Exclusive unique peptide count >10 were:
5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, chaperonin GroEL,
glutathione-binding protein GsiB, filamentous
hemagglutinin, zinc protease, ABC transporter
periplasmic amino acid-binding protein, malate
synthase G, Tol-Pal system protein TolB, pertactin
autotransporter, periplasmic solute-binding protein, aspartate-semialdehyde dehydrogenase,
outer membrane porin protein BP0840, adenosylhomocysteinase, malate dehydrogenase,
BrkA autotransporter, exported solute binding
protein, N-acetyl-gamma-glutamyl-phosphate
reductase. Experimental media CAH B. pertussis secretome included two acellular vaccine
components, but very low pertussis toxin level.
This strain has PtxP1 promoter sequence and it
was concluded that this media must be supplemented with heptakis(2,6-di-O-methyl)-β-cyclodextrin for toxin purification to be feasible. This
is an example of a one page abstract fitting to
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The anti-inflammatory effect of Limosilactobacillus Reuteri b2 administration
No full textLimosilactobacillus reuteri demonstrates a significant
role in treating gastrointestinal diseases
through the synthesis of various health-promoting
factors. These include mucus-binding proteins,
reactive oxygen species-scavenging enzymes, antimicrobial
agents (reuterin is capable of inhibiting
the growth of a wide spectrum of microorganisms),
vitamins (folate and vitamin B12), and unique
exopolysaccharides. Different strains of L. reuteri
exhibit strain-specific anti-inflammatory effects,
influencing the expression of immune-related
factors such as IL-10 and TNF-α (PMID: 20798357;
PMID: 22207578). Furthermore, the mitigating
impact of L. reuteri strains on inflammation is confirmed
in vivo and in vitro with the implication of
an interaction between probiotics and immune
cells in the intestinal mucosa (PMID: 22207578).
Our study aimed to investigate the potential anti-
inflammatory effects of daily treatment with autochthonous
probiotic strain L. reuteri B2 (PMID:
33932415) could have an anti-inflammatory effect
on local immune response. In a 14-day experiment
with Intor Swiss: Albino mice (n=10), those treated
with L. reuteri B2 (5x106 CFU/mL, 100 μl) showed a
favorable impact on the gut’s inflammatory environment.
Histological analyses of colon samples
and intraperitoneal macrophages revealed lower
myeloperoxidase (MPO) activity, reduced production
of superoxide ions, IFNγ, IL-6, and TNFα, along
with an enhanced production of IL-10 in L. reuteri
B2 treated mice compared to untreated ones. Notably,
histopathological preparations did not show
significant differences between the groups. The
study suggests that L. reuteri B2 may be valuable
for further evaluation in managing, preventing,
and treating inflammatory bowel diseases. The
presented findings contribute to understanding
the specific anti-inflammatory effects of this strain
on the local immune response, supporting its potential
as a therapeutic agent
AI-Driven Optimization of PCL/PEG Electrospun Scaffolds for Enhanced In Vivo Wound Healing
No full textHere, an artificial intelligence (AI)-based approach was employed to optimize the production of electrospun scaffolds for in vivo wound healing applications. By combining polycaprolactone (PCL) and poly(ethylene glycol) (PEG) in various concentration ratios, dissolved in chloroform (CHCl3) and dimethylformamide (DMF), 125 different polymer combinations were created. From these polymer combinations, electrospun nanofiber meshes were produced and characterized structurally and mechanically via microscopic techniques, including chemical composition and fiber diameter determination. Subsequently, these data were used to train a neural network, creating an AI model to predict the optimal scaffold production solution. Guided by the predictions and experimental outcomes of the AI model, the most promising scaffold for further in vitro analyses was identified. Moreover, we enriched this selected polymer combination by incorporating antibiotics, aiming to develop electrospun nanofiber scaffolds tailored for in vivo wound healing applications. Our study underscores three noteworthy conclusions: (i) the application of AI is pivotal in the fields of material and biomedical sciences, (ii) our methodology provides an effective blueprint for the initial screening of biomedical materials, and (iii) electrospun PCL/PEG antibiotic-bearing scaffolds exhibit outstanding results in promoting neoangiogenesis and facilitating in vivo wound treatment.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_873
Cloning, overexpression and characterization of a thermostable endo-1,4-beta-xylanase from Anoxybacillus vranjensis ST4
No full textThis research deals with the characterization of a thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus vranjensis ST4, a thermophilic bacterium isolated from Vranjska Banja hot spring, Serbia. The enzyme shows a high degree of identity with the same type of enzyme from other species of the genera Anoxybacillus (97%), Geobacillus (74%) and Paenibacillus (65%). The gene for endo-1,4-beta-xylanase from the thermophilic strain ST4 was cloned into the pQE_Ek expression vector and successfully expressed and purified from the Escherichia coli M15[pREP4]. The study encompasses recombinant production, purification, and the comprehensive characterization of the enzymatic properties of endo-1,4-beta-xylanase. This is the first successful overexpression, purification and characterization of a recombinant thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus. With a monomeric structure of 38.7 kDa, the enzyme demonstrates peak activity at 70°C and pH 6.5. Notably, it exhibits remarkable stability across a wide pH range and at high temperatures, rendering it suitable for diverse industrial applications. Investigation into the enzyme’s kinetic parameters, substrate specificity, and its ability to degrade xylan into high-energy value products further enhances understanding of its biotechnological potential. These findings underscore the significance of thermophilic bacteria and their thermostable enzymes in various industrial processes