Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein
Development of SARS-CoV-2 N-protein specific capture ELISA
Аутори
Radomirović, Mirjana Ž.Simović, Ana
Udovički, Božidar D.
Krstić-Ristivojević, Maja
Sabljić, Ljiljana Z.
Lukić, Ivana D.
Glamočlija, Sofija Đ.
Ćujić, Danica R.
Gnjatović, Marija Lj.
Stojanović, Marijana M.
Stanić-Vučinić, Dragana
Radosavljević, Jelena
Ćirković-Veličković, Tanja
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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 mi...krotitarske 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.
Извор:
58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings, 2022, 65-66Издавач:
- Belgrade : Serbian Chemical Society
Финансирање / пројекти:
Напомена:
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - CONF AU - Radomirović, Mirjana Ž. AU - Simović, Ana AU - Udovički, Božidar D. AU - Krstić-Ristivojević, Maja AU - Sabljić, Ljiljana Z. AU - Lukić, Ivana D. AU - Glamočlija, Sofija Đ. AU - Ćujić, Danica R. AU - Gnjatović, Marija Lj. AU - Stojanović, Marijana M. AU - Stanić-Vučinić, Dragana AU - Radosavljević, Jelena AU - Ćirković-Veličković, Tanja PY - 2022 UR - http://cherry.chem.bg.ac.rs/handle/123456789/5361 AB - 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. AB - 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. PB - Belgrade : Serbian Chemical Society C3 - 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings T1 - Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein T1 - Development of SARS-CoV-2 N-protein specific capture ELISA SP - 65 EP - 66 UR - https://hdl.handle.net/21.15107/rcub_cherry_5361 ER -
@conference{ author = "Radomirović, Mirjana Ž. and Simović, Ana and Udovički, Božidar D. and Krstić-Ristivojević, Maja and Sabljić, Ljiljana Z. and Lukić, Ivana D. and Glamočlija, Sofija Đ. and Ćujić, Danica R. and Gnjatović, Marija Lj. and Stojanović, Marijana M. and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Ćirković-Veličković, Tanja", year = "2022", abstract = "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.", publisher = "Belgrade : Serbian Chemical Society", journal = "58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings", title = "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein, Development of SARS-CoV-2 N-protein specific capture ELISA", pages = "65-66", url = "https://hdl.handle.net/21.15107/rcub_cherry_5361" }
Radomirović, M. Ž., Simović, A., Udovički, B. D., Krstić-Ristivojević, M., Sabljić, L. Z., Lukić, I. D., Glamočlija, S. Đ., Ćujić, D. R., Gnjatović, M. Lj., Stojanović, M. M., Stanić-Vučinić, D., Radosavljević, J.,& Ćirković-Veličković, T.. (2022). Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings Belgrade : Serbian Chemical Society., 65-66. https://hdl.handle.net/21.15107/rcub_cherry_5361
Radomirović MŽ, Simović A, Udovički BD, Krstić-Ristivojević M, Sabljić LZ, Lukić ID, Glamočlija SĐ, Ćujić DR, Gnjatović ML, Stojanović MM, Stanić-Vučinić D, Radosavljević J, Ćirković-Veličković T. Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings. 2022;:65-66. https://hdl.handle.net/21.15107/rcub_cherry_5361 .
Radomirović, Mirjana Ž., Simović, Ana, Udovički, Božidar D., Krstić-Ristivojević, Maja, Sabljić, Ljiljana Z., Lukić, Ivana D., Glamočlija, Sofija Đ., Ćujić, Danica R., Gnjatović, Marija Lj., Stojanović, Marijana M., Stanić-Vučinić, Dragana, Radosavljević, Jelena, Ćirković-Veličković, Tanja, "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein" in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings (2022):65-66, https://hdl.handle.net/21.15107/rcub_cherry_5361 .