Magnetic Beads Quantitative-PCR Based Method For Evaluating Activities Of SARS-CoV-2 Neutralization Antibodies

The Coronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is an unprecedented health crisis facing the world,which has not completely ended yet.Establishing effective immunity against the virus through vaccination was one of the key measures to finally overcome the epidemic.However,due to the different health status of individuals,the rapid virus evolution,the vaccine types,and the fading of antibody titers,the individual vaccination effect is usually variable.In post-epidemic era,the neutralizing activity and persistence of antibodies induced by vaccines are the important markers of long-term protection.Therefore,we here developed a new method for evaluating the neutralizing activity of SARS-CoV-2 antibodies in vitro based on the mechanisms of interactions between the virus and host cells.First,a recombant probe of RBM-DNA for quantitative PCR was prepared by covalently coupling the receptor binding motif(RBM)of the S protein to a DNA amplification reporter.Meanwhile,we constructed a pentamer RBM-binding protein 2B-LCB1 which was coupled with magnetic beads as a rapid capturer(Mag-LCB1)of RBM-DNA.Finally,the interaction between RBM-DNA probe and Mag-LCB 1 capturer was quantitatively detected by qPCR,which was further applied to evaluate the neutralization activity of SARA-CoV-2 antibodies.The main research contents and conclusions are as follows.(1)Preparation of antigenic protein RBM-C3 and capture protein 2B-LCB1.E.coli strains for expressing RBM-C3 recombinant protein and 2B-LCB1 recombinant protein were successfully constructed.By optimizing fermentation conditions,the yield of RBM-C3 recombinant protein was 9 mg·L-1 and the yield of 2B-LCB1 recombinant protein was 5 mg·L-1(2)Activity verification of antigenic protein RBM-C3.ELISA analysis showed that RBM-C3 protein prepared through purification and renaturation has the binding ability with a commercial RBD neutralizing antibody and 2B-LCB1 with IC50 of 3.76 μM and 0.215 μM,respectively,which met the requirements for the core antigen of the hybrid probe to develop RBDn-PCR in this paper.(3)Preparation of probe element RBM-DNA and capture element Mag-LCB1.The hybrid probe of Mag-LCB1 was successfully prepared by coupling RBM-C3 with the amplification DNA.The capturing element of the hybrid probe,i.e,Mag-LCB1,was successfully constructed by immobilizing the 2B-LCB1 on the COOH-functionalized magnetic beads.Quantitative detection of the interaction between Mag-LCB1 and RBM-DNA was achieved by qPCR,and the method was termed as RBDn-PCR.The RBDn-PCR can detect a commercial RBD neutralizing antibody with the limit of detection(LOD)4.05μg·mL-1.(4)RBDn-PCR was used to evaluate the activity of RBD neutralizing antibody in serum samples.The RBDn-PCR was validated by detecting 86 serum samples from vaccinated individuals.Among those samples,1.16%showed the lowest neutralizing activitiy,while 4.76%showed the highest neutralizing activitiy.The samples with moderate neutralizing activity accounted for the highest proportion(53.6%).These data with normal distribution indicated that the RBDn-PCR was able to distinguish the different neutralizing activity from individuals.