Development of SARS-CoV-2 N-protein specific capture ELISA

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.Poster: [https://cherry.chem.bg.ac.rs/handle/123456789/5362

Development of SARS-CoV-2 N-protein specific capture ELISA

Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja zasuzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostimaELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studijeje bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih serumaza rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjamapsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testaproizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima ize evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumivisokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog zaovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adheriranina dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli itekoncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu zakvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jimpoliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe ujespektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikacijuN-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototipELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekcijuN-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 zakvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.The accurate diagnosis of people with suspected infection with the SARS-CoV-2 isessential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can bedetected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens inbiological 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 forroutine measurement of the concentration of SARS CoV-2 nucleocapsid protein usingabsorbance measurement in a standard 96-well microtiter plate. For the purposes of the testdevelopment, recombinant N protein was produced and used for the production of miceand rabbit antisera. Produced antisera were purified and titer was determined. High-affinitypolyclonal N-protein specific antisera were used for N-protein specific ELISA testdevelopment. The test is based on mice polyclonal sera adhered to microtiter plate bottomfor the capture of the N protein from the specimen. Various concentrations of therecombinant 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 andanti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.We have successfully developed the prototype ELISA for the quantification of N-proteinwith the detection limit being in the range of ng/mL. The average LOD value for theprototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for thedetection of N-protein with affinity and specificity similar to, or better than commercialantibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence forquantification of the N-protein in protein-rich samples, similar to human sera.Abstract: [https://cherry.chem.bg.ac.rs/handle/123456789/5361

Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species

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

Multiaxial determination of the resistance to creasing of clothing wool fabrics

In this paper, values of deformation components (elastic, viscoelastic, plastic) of clothing wool fabrics by measuring the crease recovery angle in various directions to the warp direction (0°-warp, 30°, 45°, 60°, and 90°-weft) were determined. The size as well as the change of deformation component from warp to weft direction was presented through polar diagrams. On the basis of the results of investigation it is possible to conclude that all investigated fabrics (plain, 2/2 twill, cross twill), regardless of the biaxiality concerning quickly reversible (elastic) deformation, tend toward isotropy in total reversible deformation (elastic + viscoelastic). Concerning the plastic deformation value, mentioned investigated fabrics also express tendency toward isotropy

Some Properties of Antimicrobial Coated Knitted Textile Material Evaluation

The aim of this work was to investigate the antimicrobial activity of knitted fabric intended for medical purposes as well as the influence of antimicrobial treatment on the compression work, volume resistivity, water sorption. and water retention of the examined material. Polyamide/elastane plain jersey knitted fabric was treated with a polymer matrix of chitosan gel in which either gentamicin sulfate or autochthonous essential oil of Picea abies was incorporated as an active substance. The treated fabrics showed a wide range of antimicrobial activity on microorganisms (Staphilococcus aureus, Escherichia coli, Klebsiella and Candida albicans ) and therefore may have various medical applications. However, antimicrobial treatment changed the mechanical and physical-hygienic properties of the knitted fabric. The antimicrobial coatings decreased the total, elastic and irreversible compression work, water sorption, and volume resistivity, and increased the water retention of the fabrics. Antimicrobial treatment with chitosan and gentamicin sulfate produced a knitted fabric with good antimicrobial properties. However, if good mechanical and physical-hygienic properties are required, antimicrobial treatment with chitosan and essential oil of Picea abies has the advantage over treatment with gentamicin sulfate

Ispitivanje kompresione sposobnosti antibakterijski obrađenog medicinskog tekstilnog materijala

In this paper the possibility of the application of textile knitted material as a compression material from the aspect of the antibacterial treatment as well as the human body constitution was investigated. PA 6.6/Elastane weft knitted fabric, treated with gentamicin sulfate, was used as a suitable experimental material. Compression ability of investigated knitted fabric was examined through the value of pressure which material generates during the application on the certain part of the human body. Constitution type of the female leg, which might be slim, normal and plump, was the base on which the influence of the human body constitution on pressure distribution performance was monitored. The value of pressure was determined by the application of the La-Place equation. Results of the imposed experiment showed that pressure, which textile material generates by wrapping the human model's leg, increases, in a certain extent, due to the applied antibacterial treatment. Keeping in mind that percentage deviation of the pressure from the recommended value is in the limits of the al lowed exception it was established that investigated knitted fabric has satisfied compression ability. Observed knitted fabric might be successfully used in medical purposes as a compression material concerning all three constitution types of leg, before as well as after antibacterial treatment, rather widthwise than lengthwise.U radu je ispitivana mogućnost primene pletenog tekstilnog materijala za kompresionu terapiju sa aspekta antibakterijske obrade pletenine i anatomske grade ljudskog tela. Za realizaciju eksperimenta upotrebljena je glat desno-leva potkina pletenina sirovinskog sastava PA 6.6/Elastan, koja je obrađena gentamicinsulfatom. Kompresiona sposobnost ispitivane pletenine je praćena preko vrednosti pritiska koji materijal proizvodi pri aplikaciji na određeni deo tela tokom perioda nošenja. Uticaj grade ljudskog tela na raspodelu pritiska posmatran je na osnovu konstitucionog tipa ženske noge koji može biti vitki, normalni i puniji. Vrednost pritiska je određena primenom La Placeove jednačine. Rezultati sprovedenog eksperimenta su pokazali da antibakterijska obrada u izvesnoj meri povećava vrednost pritiska koji materijal stvara pri obavijanju noge ispitanika. Takođe je ustanovljeno da ispitivana pletenina ima zadovoljavajuću kompresionu sposobnost, s obzirom da se procentualno odstupanje pritiska od preporučene vrednosti nalazi u granicama dozvoljenog odstupanja. Posmatrana pletenina se mole upotrebiti u medicinske svrhe kao kompresioni materijal za sva tri konstituciona tipa noge i pre i posle antibakterijske obrade, pre po širini nego po dužini

Antimikrobna biološki-aktivna vlakna za medicinsku namenu

The use of textile materials in medicine has a long tradition. An important field of application of textiles in medicine is in wound care and prevention of chronic wounds, for which bandages and wound dressings are used. Modern medicine demands textile materials with special properties. The most important are biologically active fibers, and among them antimicrobial fibers which are able to kill or inhibit the growth of bacteria, fungi and/or other pathogenic microorganisms. Due to their high efficacy, antimicrobial fibers have become necessary in prevention and therapy, especially in festering wound healing and prevention of chronic wounds. Among all biologically active fibers and biomedical textile materials antimicrobial fibers are mostly used, mainly because of their ability for controlled and long-term releasing of antimicrobial agents and their stability against inactive factors, as well as their simple and easy application. The scope of this review focuses on properties of antimicrobial fibers and mechanisms of their antimicrobial activity, as well as physicochemical bases of antimicrobial fibers obtaining and test methods used to confirm their antimicrobial activity. Also, representative pathogenic microorganisms are listed. Antimicrobial fibers of natural and synthetic origin are reviewed, as well as advances and future trends in their development.Medicinski tekstilni materijali se veoma dugo i široko koriste u medicinske svrhe. Posebno važno mesto za- uzimaju kao materijali za previjanje, naročito u hirurgiji i lečenju gnojnih rana. Savremena medicina zahteva tekstilne materijale sa specijalnim svojstvima. Najznačajniji među njima su biološki-aktivna vlakna, a u okviru njih antimikrobna vlakna, koja poseduju sposobnost da uništavaju ili sprečavaju razvoj bakterija, gljivica i druge patogene flore. Zbog svoje visoke efektivnosti antimikrobna vlakna zauzimaju značajno mesto u profilaksi i terapiji, posebno gnojnih procesa i rana. Njihova sposobnost da kontrolisano i prolongirano otpuštaju lekoviti antimikrobni preparat, da su stabilna na dejstvo inaktivirajućih faktora, da se jednostavno i lako apliciraju stavlja ih na prvo mesto medu biološki-aktivnim vlaknima i biomedicinskim tekstilnim materijalima. U radu su obrađena svojstva antimikrobnih vlakana, reprezentativni patogeni mikroorganizmi i mehanizam antimikrobne aktivnosti biološki-aktivnih vlakana. Takođe, obrađene su fizičko-hemijske osnove dobijanja antimikrobnih vlakana i metode njihovog karakterisanja. Dat je pregled pojedinačnih antimikrobnih vlakana prirodnog i sintetizovanog porekla kao i savremeni trendovi i budući pravci njihovog daljeg razvoja

Physical-mechanical phenomenon of woven and non-conventional materials

The basic idea of this project is in developing series of methods for monitoring structural parameters, as well as establishing their relation with the behaviour of woven and non-woven materials. Establishing the connection among structural parameters of material (constructive elements, their position and distribution, construction) and their behavior during special conditions of usage, through monitoring the phenomenon of physical-mechanical characteristics and resistance mechanisms, contributes to establishing the relation for developing complex methodology of projecting textile surfaces. Investigation of textile complexes as porous and flexible systems represents the basis for establishing functional dependence "fibre-textile complex-final product" type. This is possible to achieve through the analysis of textile surface state and its topography, as well as constructive solutions and physical-mechanical characteristics. Analytic investigation of characteristics, such as flexibility and resistance to various strains, was conducted on woven fabrics and non-woven materials of various constructions aimed at establishing their basic models. The main focus is on mechanical characteristics, especially elastic properties under action of various loads (tensile, bending, pressure). Relaxation processes and recovery kinetics are particularly investigated. Making the connection between structure and mechanical characteristics enables the projection of the expected or desired behavior of woven fabrics. Besides, establishing criteria for estimation of the total elasticity, as the magnitude of quality index and its valorization, represents the possibility for establishing objective methods of woven and nonwoven materials of functional characteristics

Alterations in the reptilian fauna of Serbia: Introduction of exotic and anthropogenic range expansion of native species

This study describes cases of introductions of exotic species and range expansion of native species in Serbia. The four most important introductions are the alien, invasive Trachemys scripta elegans and native Testudo hermanni, Mediodactylus kotschyi and Podarcis muralis which expanded their ranges. Trachemys scripta elegans should be of special concern, since introductions of this species can have numerous negative effects on local ecosystems. Incidental introductions were documented for two alien (Pelodiscus sp. and Hemidactylus turcicus) and two native species (Podarcis tauricus and Vipera ammodytes). The greatest number of introductions is concentrated around urban areas, especially around the cities of Belgrade and novi Sad. Increased traffic, trading and cargo shipment, as well as a large number of people who keep exotic animals as pets, make large urban areas susceptible to accidental and deliberate introductions of amphibians and reptiles. on the other hand, anthropogenic alteration and degradation of habitats, along with increased urbanization, can provide corridors for the range expansion of native species

Mathematical Model of Gentamicin Sulfate Release from a Bioactive Textile Material as a Transdermal System Under In Vitro Conditions

A mathematical model was developed to estimate the release of gentamicin sulfate from a bioactive textile material as a transdermal system for wound dressing. The gentamicin sulfate released from the antibiotic/chitosan hydrogel complexes was measured in vitro by the Franz diffusion cell technique. The diffusive transport of gentamicin sulfate through three connected compartments, that is, chitosan hydrogel, membrane, and solution, was considered by the formulation of a model based on Fick's second law. Initially, the total amount of gentamicin sulfate was placed within an already swollen chitosan hydrogel. The value of the diffusivity coefficient of the drug through the chitosan hydrogel was calculated for every initial amount of the active substance. For the initial concentration of gentamicin sulfate, which was lower than 2.81 x 10(4) mu g/mL, the diffusion coefficient was approximately constant. A higher amount of gentamicin sulfate in the hydrogel reduced its own transport as a consequence of an increase in the intensity of the interaction field between the molecules of gentamicin sulfate. The model provides the possibility of optimizing the process of drug release by ensuring a compromise between a higher value of the diffusivity coefficient and a desirable amount of gentamicin sulfate and a constant concentration within the solution over 48 h