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Acuña Intrieri, M. E  
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Deriane, M.A  
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Miller, C.  
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Czibener, Cecilia  
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Correa, E.  
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Cragnaz, L.  
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Guerra, L.  
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Rodriguez, S.  
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Goldbaum, F.A.  
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Seigelchifer, M.  
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Comerci, Diego José  
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Montagna, Georgina Nuri  
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Cerutti, Maria Laura  
dc.date.available
2022-03-22T11:46:13Z  
dc.date.issued
2021  
dc.identifier.citation
Development of COVID-19 monoclonal antibodies and recombinant proteins as reagents for biomedical research and diagnostic test; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research y XVI Annual Meeting of the Argentinean Society for General Microbiology; Virtual; Argentina; 2021; 1-1  
dc.identifier.issn
0327-9545  
dc.identifier.uri
http://hdl.handle.net/11336/153723  
dc.description.abstract
Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293 suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routinary lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminium salts (RBD/Al) and the other with RBD emulsified in Freunds adyuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.  
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application/pdf  
dc.language.iso
eng  
dc.publisher
Sociedad Argentina de Bioquímica y Biología Molecular  
dc.rights
info:eu-repo/semantics/openAccess  
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/  
dc.subject
MONOClONAL ANTIBODY  
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RBD  
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HEK  
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COVID-19  
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Bioquímica y Biología Molecular  
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Ciencias Biológicas  
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CIENCIAS NATURALES Y EXACTAS  
dc.title
Development of COVID-19 monoclonal antibodies and recombinant proteins as reagents for biomedical research and diagnostic test  
dc.type
info:eu-repo/semantics/publishedVersion  
dc.type
info:eu-repo/semantics/conferenceObject  
dc.type
info:ar-repo/semantics/documento de conferencia  
dc.date.updated
2022-03-14T20:43:42Z  
dc.journal.pagination
1-1  
dc.journal.pais
Argentina  
dc.journal.ciudad
Mendoza  
dc.description.fil
Fil: Acuña Intrieri, M. E. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina  
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Fil: Deriane, M.A. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina  
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Fil: Miller, C.. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina  
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Fil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina  
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Fil: Correa, E.. No especifíca;  
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Fil: Cragnaz, L.. No especifíca;  
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Fil: Guerra, L.. No especifíca;  
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Fil: Rodriguez, S.. No especifíca;  
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Fil: Goldbaum, F.A.. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina  
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Fil: Seigelchifer, M.. No especifíca;  
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Fil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina  
dc.description.fil
Fil: Montagna, Georgina Nuri. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina  
dc.description.fil
Fil: Cerutti, Maria Laura. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/http://www.saib.org.ar/index.php?q=node/562  
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Nacional  
dc.type.subtype
Congreso  
dc.description.nombreEvento
LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research y XVI Annual Meeting of the Argentinean Society for General Microbiology  
dc.date.evento
2021-11-01  
dc.description.ciudadEvento
Virtual  
dc.description.paisEvento
Argentina  
dc.type.publicacion
Journal  
dc.description.institucionOrganizadora
Sociedad Argentina de Investigación Bioquímica y Biología Molecular  
dc.description.institucionOrganizadora
Asociación Civil de Microbiología General  
dc.source.revista
Biocell  
dc.date.eventoHasta
2021-11-05  
dc.type
Congreso