Development And Application Of A Highly Sensitive Method For The Detection Of Neutralizing Antibodies To SARS-CoV-2

Accurate detection of neutralizing antibodies against SARS-CoV-2 is critical for determining the level of immune response following natural infection or vaccination.Several competitive-binding methods have been developed for SARS-CoV-2 neutralizing antibody detection,which quantitatively measure the neutralizing activity of antibodies by detecting the inhibition of binding between the spike(S)protein of the virus and its cellular receptor,angiotensin converting enzyme 2(ACE2),in serum samples.These methods are rapid and cost-effective alternatives to virus-based neutralization assays.To better understand the competitive-binding strategy for detecting SARS-CoV-2 neutralizing antibodies,we established a magnetic bead/flow cytometry-based method and evaluated the performance of six different combinations of immobilized ACE2 or S1/RBD proteins and soluble Fc or His-tagged S1/RBD or ACE2 proteins.These combinations included:(1)immobilized ACE2/soluble Fc-tagged S1 subunit of S protein(i ACE2/S1-Fc),(2)immobilized ACE2/soluble Fc-tagged receptor binding domain of S protein(i ACE2/RBD-Fc),(3)immobilized S1 protein/soluble Fc-tagged ACE2 protein(i S1/ACE2-Fc),(4)immobilized S1 protein/soluble His-tagged ACE2 protein(i S1/ACE2-His),(5)immobilized RBD protein/soluble Fc-tagged ACE2 protein(i RBD/ACE2-Fc),and(6)immobilized RBD protein/soluble His-tagged ACE2 protein(i RBD/ACE2-His).The detection performance of these six models was evaluated using three functionally characterized SARS-CoV-2 monoclonal neutralizing antibodies,convalescent plasma from COVID-19 patients,and immune sera from vaccinated individuals.The i ACE2/RBD-Fc,i ACE2/S1-Fc,and i S1/ACE2-His models showed good detection specificity,with the i ACE2/RBD-Fc model having the highest detection sensitivity,which was significantly higher than that of a commercial surrogate virus neutralization test(s VNT)ELISA kit.Further studies revealed that the sensitivity and specificity of the competitive binding based assays for detection of SARS-CoV-2 neutralizing antibodies were affected by the tag of the ACE2 fusion protein,the type of spike protein,and the method of measuring the binding between the spike protein and ACE2 protein.In addition,the i ACE2/RBD-Fc model was able to effectively detect the dynamic changes in neutralizing antibody activity against both the original SARS-CoV-2 prototype and its Omicron variant in sera from individuals who received multiple doses of inactivated COVID-19 vaccines.The results of this model showed a significant correlation with those obtained using neutralization assays based on live and pseudo viruses.These findings suggest that the i ACE2/RBD-Fc model has the potential to become a highly sensitive,specific,and versatile method for SARS-CoV-2 neutralizing antibody detection in clinical settings.The insights gained from this study may aid in the design and development of competitive-binding neutralizing antibody assays for SARS-CoV-2 and other viruses.