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

JUAN IGNACIO MARFIA; ADRIANA VICTORIA SABLJIC; SILVINA SONIA BOMBICINO; ALDANA TRABUCCH; RUBEN F IACONO; IGNASIO SMITH; GREGORIO MC CALLUM; FEDERICO J. WOLMAN; ALEXANDRA MARISA TARGOVNIK; JOAQUÍN POODTS; MATÍAS FINGERMANN; ADOLFO ROOT; MARCELO RODRIGUEZ FERMEPIN; LUCÍA GALLO VAULET; LEONARDO GABRIEL ALONSO; MARÍA VICTORIA MIRANDA; SILVINA NOEMÍ VALDEZ

Congreso; Reunión anual de sociedades de biociencias; 2022

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 wasproduced at high scale in horse. This assay is aimed for diagnosisof COVID-19 and quantification of RBD and S protein from any production process. Materials and methods: Equine polyclonal anti-Santibodies were obtained by immunization of one mixed-breed 4 to10 years-old, 300 to 450 kg horse. The purified antibodies were incubated with a 20-fold molar excess of sulfo-NHS-biotin. Free biotinwas removed on a PD-10 desalting column. The ELISA was basedon the capture of the antigen present in samples or calibration curveby equine anti-S antibodies immobilized in the solid phase. BoundRBD or S protein was detected by the addition of antibodies anti-S-biotin followed by Streptavidin-Horseradish Peroxidase. Twentyhuman samples from the respiratory tract were analyzed in parallelby rRT-PCR and by ELISA. Additionally, recombinant S protein orRBD both expressed in baculovirus/ insect larvae were detected andquantified. Results: Out of the 20 patient samples, 15 were positivefor rRT-PCR and 5 were negative. When a cut-off value of 0.1 ODwas established, 10 out of the 15 positive samples were also positive by our developed ELISA (sensitivity: 67%). Moreover, the testshowed a lineal response for concentrations of 2.4 to 35x10-11 M and1 to 20x10-11 M for RBD and S protein, respectively. So, this assayallows the quantification not only in biological samples but also inproduction processes of both recombinant proteins. Conclusion:Our results demonstrate that the method developed herein, basedon detection of RBD and S protein of SARS-CoV-2, is useful as analternative and rapid screening method for diagnosis of COVID-19in low or medium complexity laboratories. Moreover, this assay canalso be applied for the quantification of recombinant RBD or S protein