Human health risk assessment due to heavy metals in surface soil surrounding “Nikola Tesla A” thermoelectric power plant

PP) represent one of major sources of environmental pollution [1]. Coal combustion leads to accumulation of heavy metals (HMs) in combustion by-products whose disposal and atmospheric emission are main pathways for dispersion of HMs in the soil surrounding TEPPs. HMs from soil may reach human body via variety of pathways, therefore the resident population near TEPPs should be considered to be continuously exposed to soil and coal combustion residuals contaminated by HMs. The TEPP “Nikola Tesla A” is the largest TEPP in Serbian electric power industry. It is located near Obrenovac, (35 km from Belgrade), in the area identified as the Serbian region most threatened by pollution from coal mining and coal combustion.The aim of the present study was to assess carcinogenic and non-carcinogenic health hazard for residents associated with HMs in soil. The potential human health riskwas estimated for exposures to minimal, mean and maximal total measured concentration of selected HMs – Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn. In case of Cr, 6:1 ratio of Cr(III):Cr(VI) was applied as recommended by US EPA. Surface soil samples (10 cm depth) were collected at 30 locations distributed 1, 2, 4, 6, 8, and 10 km to the west, southwest, south, southeast, and east directionfrom the TEPP “Nikola Tesla A”. Total concentrations of HMs were measured by atomic absorption spectrometer. The US EPA risk assessment model [2] was exploited for risk calculation taking into account ingestion of soil, inhalation of resuspended soilparticles and dermal exposure to HMs in soil. The cancer risk was evaluated through the excess lifetime cancer risk – ELCR, and non-carcinogenic risk was expressed as the hazard quotient – HQ. According to US EPA, the cancer risk below 106 is considered to be negligibly small, and risk of 104 to be sufficiently large that remediation is desirable. Cancer risk between 106 and 104 are generally considered acceptable [3].The value of HQ should be less than unity to consider risk from systemic toxicity negligible. The total cancer risk, ELCRtotal, is calculated as a sum of all ELCR for all HMs and all exposure routes considered. The overall non-carcinogenic risk is expressed as hazard index, HI, equal to the sum of all HQ for all HMs and all accounted exposure routes[2]. Risk assessment from non-carcinogenic effects showed that risk from ingestion of soil particles by children and adults comprises almost whole HI. Dermal risk existed only for exposition to Cd in soil, and it was negligible for both categories (4 × 109 to 3 × 103 ). Risk arose from inhalation exposure was not respective because calculated HIwas so benevolent with maximal value of 1 × 108 for both children and adults. Although none of HQ for any single HM was above the reference value of one, aggregate HI for children fell in the range from 1.04 to 2.60 with a mean value of 1.79. Cobalt (0.47 < HI <1.00), Fe (0.42 < HI <0.94) and Mn (0.11 < HI <0.44) were identified as contaminants of most concern. Among HMs measured, only Cd, Co, Cr(VI), Ni and Pb are recognized as human cancerogens [2]. The ELCRtotalfell in the range from 1 × 105 to 5 × 105 . Ingestion of soil contributed the largest to the ELCRtotal, followed by insignificant contribution from inhalation. There was no risk induced by dermal exposure. According to Institute of Public Health of Serbia, the standardized cancer incidence for in 2014 for the City of Belgrade (where the municipality of Obrenovac belongs) was 2.60 × 103 for males and 2.15 × 103 for females [4]. These values are very high in comparison to the risk assessed in this study; therefore, the risk provoked by exposure to HMs in soil made portion of the real cancer risk that was completely insignificant. The estimated carcinogenic risk in this research was in the acceptable range. Estimated non-carcinogenic risk suggests that adults are not endangered due to HMs in soil, while children population is under elevated risk from deleterious health effects. Ingestion of soil was identified as a primary pathway of HMs harming to human health.8th Symposium Chemistry and Environmental Protection : May 30 - June 1, Kruševac, 2018

SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children

RT-PCR remains the primary method of diagnosing SARS-CoV-2. Serological diagnosis of COVID-19 is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false negative when antibody concentrations fall below detection limit. Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. Antigen-based tests show high specificity, but sensitivity is relatively low. For development and further improvement of both serological and antigen-based tests, availability of recombinantly producted SARS CoV-2 antigens is needed and during the periods of high demend in the pandemic, it was a limited factor both for research and diagnostic tests production by national producers of tests. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of beta-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Application of serological ELISA for seropositivity detection in 200 allergic children in comparison to non-allergic did not show a bias towards any of the tested groups. Furthermore, we have optimized production of rfNP at a large scale to raise specific antibodies to N-protein in rabbit and mice and develop an in house ELISA for quantification of N protein in biological fluids. Test was clinically validated in 200 PCR positive patients and showed a sensitivity of around 50%

Serological Elisa test development at the INEP Institute

The COVID-19 diagnostic tools are categorized into two main groups of Nucleic Acid (NA)- based and protein-based tests. To date, nucleic acid-based detection has been announced as the gold-standard strategy for coronavirus detection; however, protein-based tests are promising alternatives for rapid and large-scale screening of susceptible groups. During the first months, no rapid and reliable detecting tool was readily available to adequatly respond to the requirement of massive testing. The aim was to develope cost-effective, sensitive and rapid screening mechanisms for the detection of immune response to SARS-CoV2 (which causes COVID-19), based on the principle of ELISA. The institute INEP has developed tests for detection of IgM and IgG SARS-CoV-2 specific antibodies based on S and N proteins of virus intended to monitor different phases of natural infection, ELISA test for IgG detection in naturale infection based on the use of exclusively domestic components of the ELISA kit (including proteins produced in Serbia, Faculty of Chemistry) and a test specifically designed to monitor the effects of immunization (determination of IgG antibodies specific for the RBD domain of S protein). The tests were independently validated at the relevant laboratories in the country and abroad, and compared to an FDA/WHO approved tests of a major test producers. All testing for validation was carried out on samples collected before COVID19 (negative controls), PCR confirmed COVID19 samples (positive controls) and potencialy cross-reactive samples (other pathogens and autoimmune diseases). The antibody tests showed high levels of sensitivity and specificity and extremely low background noises. The kits are extremely stable, have a shelf life of 1 year and opened kits are usable up to 3 months at storing temperatures of 5°

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

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

Content of the potentially harmful elements in soil around the major coal-fired power plant in Serbia: relation to soil characteristics, evaluation of spatial distribution and source apportionment

The concentrations and spatial distribution of nine potentially harmful elements (PHEs), namely Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn, and their relation to soil properties were investigated in thirty soil profiles (0-50 cm depth) sampled around the largest Serbian coal-fired power plant (CFPP) "Nikola Tesla A." Soil properties were determined following standard procedures, and total contents of PHEs were analyzed by atomic absorption spectrometer. Concentrations of Cd, Co, Fe, Mn, Pb and Zn were the highest in soil profiles sampled 1 km away from the CFPP, concentrations of Ni and Cu gradually increased up to 4 km, and the highest Cr concentrations were measured in samples taken 6 km away from the CFPP. The highest concentration of PHEs analyzed, except Mn, corresponded with predominant wind directions. Depth did not show significant impact on distribution of any PHEs investigated. Among soil properties, the total organic carbon showed the closest relationship with the PHEs. Data were processed by a principal component analysis which enabled distinguishing anthropogenic from natural influences on soil properties and PHE contents. Although the impact of CFPP operations is obvious, assets of principal component analysis did not allow clear distinction of CFPP's contribution from parent material in enrichment of PHE contents in the soil in the study area