NOVEL QUANTITATIVE ENZYME LINKED IMMUNOSORBENT ASSAY TO DETECT RBD AND SPIKE PROTEIN OF SARS-COV-2 USEFUL FOR DIAGNOSIS OF COVID-19

MARFÍA, JUAN IGNACIO; SABLJIC, ADRIANA V.; BOMBICINO, SILVINA S.; TRABUCCHI, ALDANA; IACONO, RUBÉN F.; SMITH, IGNACIO; MC CALLUM, GREGORIO JUAN; WOLMAN, FEDERICO JAVIER; TARGOVNIK, ALEXANDRA MARISA; POODTS, JOAQUÍN; FINGERMANN, MATÍAS; DE ROODT, ADOLFO; RODRIGUEZ FERMEPIN, MARCELO; GALLO VAULET, LUCÍA; ALONSO, LEONARDO GABRIEL; MIRANDA, MA. VICTORIA; VALDEZ, SILVINA N.

Mar del Plata

Congreso; LXX REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2022

Sociedad Argentina de Inmunología

Herein we describe a novel enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 antigen detection employing a high affinity polyclonal serum to recombinant Spike (S) protein, which was produced at high scale in horse. This assay is aimed for diagnosis of COVID-19 and quantification of RBD and S protein from any production process.Materials and methods: Equine polyclonal anti-S antibodies were obtained by immunization of one mixed-breed 4 to 10 years-old, 300 to 450 kg horse. The purified antibodies were incubated with a 20-fold molar excess of sulfo-NHS-biotin. Free biotin was removed on a PD-10 desalting column. The ELISA was based on the capture of the antigen present in samples or calibration curve by equine anti-S antibodies immobilized in the solid phase. Bound RBD or S protein was detected by the addition of antibodies anti-S-biotin followed by Streptavidin-Horseradish Peroxidase. Twenty human samples from the respiratory tract were analyzed in parallel by rRT-PCR and by ELISA. Additionally, recombinant S protein or RBD both expressed in baculovirus/ insect larvae were detected and quantified.Results: Out of the 20 patient samples, 15 were positive for rRT-PCR and 5 were negative. When a cut-off value of 0.1 OD was established, 10 out of the 15 positive samples were also positive by our developed ELISA (sensitivity: 67%). Moreover, the test showed a lineal response for concentrations of 2.4 to 35x10-11 M and 1 to 20x10-11 M for RBD and S protein, respectively. So, this assay allows the quantification not only in biological samples but also in production processes of both recombinant proteins.Conclusion: Our results demonstrate that the method developed herein, based on detection of RBD and S protein of SARS-CoV-2, is useful as an alternative and rapid screening method for diagnosis of COVID-19 in low or medium complexity laboratories. Moreover, this assay can also be applied for the quantification of recombinant RBD or S protein.