ELISA Kits ACE2-RBD Neutralization Assay

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was first identified amid an outbreak of respiratory illness cases in Wuhan City, Hubei Province, China and has since then caused a global pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA virus and belongs to the Betacoronavirus Genus, which also includes SARS CoV (2003) and MERS CoV (2012). Same as all other coronaviruses, the genome of SARS-CoV-2 (2019-nCoV) encodes the Spike/RBD protein, the envelope protein, the membrane protein, and the nucleocapsid protein or NCP. While antibodies to NCP are the first to appear in seroconversion, antibodies to Spike/RBD, involved in the ACE2 receptor binding, are produced later during the infection, are considered a marker of recovery and are known to have a neutralizing effect on SARS-CoV-2 by blocking the binding of the virus to ACE2 receptor. ACE2-RBD Neutralization Assay is an ELISA Kit, used for the determination of the neutralizing activity of anti SARS-CoV-2 antibodies by ACE2-RBD binding inhibition in human serum and plasma. PRINCIPLE OF THE TEST The inhibition of the binding between ACE2 and RBD is determined by means of an ELISA carried out on plasma/sera whose antibodies neutralizing action wants to be measured. Microplates are coated with SARS-CoV-2 specific recombinant glycosylated RBD. The sample is incubated allowing anti Spike/RBD antibodies, if present, to bind to such antigen. After washing free Spike/RBD are determined by the addition of recombinant ACE2 biotinilated antigen and then in sequence of SAV-HRP. A color will be generated by TMB/H2O2 if no antibodies have bound to RBD while a strong inhibition on the color development will be observed in case antibodies to RBD have blocked the binding of the biotin-labelled ACE2 to it. The presence of such antigen on the solid phase is finally determined by the addition of SAV-HRP, which will bind to ACE2 if no neutralizing antibodies are present or not in case antibodies have blocked the coated RBD. In case the sample is diluted serially in the assay a titer can be calculated by the system providing a value of neutralization. MATERIALS AND REAGENTS Microplate Negative Control Positive Control ACE2-biotin Streptavidin-HRP Assay Diluent Chromogen/Substrate Wash buffer concentrate Sulphuric Acid Plate sealing foils Package insert PERFORMANCE Whole performances are still under evaluation considering the difficulty to evaluate in particular specimens coming from infected hospitalized patients. The assay shows a correlation with the DiaPro’s ELISA for anti Spike/RBD antibodies on positive samples > 98%. The kit is intended for “in vitro” diagnostic use only.