Development Of Monoclonal Antibodies Against SARS-CoV-2 Nucleocapsid Protein And Establishment And Application Of Competitive ELISA Method

Coronavirus disease 2019（COVID-19）,caused by Severe Acute Respiratory Syndrome Coronavirus 2（SARS-CoV-2）infection,has been reported more than 650 million confirmed cases and more than 6.6 million deaths worldwide,causing huge losses to human life,and social and economic development.There are many variants of SARS-CoV-2,of which Alpha,Beta,Gamma,Delta,and Omicron are the main variants that have spread widely.The current global epidemic strains are mainly Omicron（BA.5）,and（BA.XXB.1）.Most mutations of these variant strains occurred in the spike protein（S）,while the nucleocapsid（N）gene sequence is relatively conserved.The N protein of SARS-CoV-2 plays an important role in the replication of the virus,and high levels of N protein are present in the blood of patients early in infection.Antibodies of IgM and IgG to N protein could be detected in 7 d and 7 d～14 d after infection respectively,suggesting the good immunogenicity of N protein.N protein and its specific antibodies are important detection targets for SARS-CoV-2 infection,and the detection of antibodies to SARS-CoV-2 N protein shows higher sensitivity than S protein.In this study,the E.coli expression system and the mammalian expression system were used to express and purify the SARS-CoV-2 recombinant N protein,and monoclonal antibodies（mAbs）specifically recognizing the recombinant N protein were screened and prepared by B-cell hybridoma technology.The dominant mAb with strong specific recognition ability to SARS-CoV-2 recombinant N protein was further analyzed and screened to establish competitive ELISA method for detection of antibodies to N protein of SARS-CoV-2.The competitive ELISA detection method is initially used for human and different animal serological detection,which providing an efficient and rapid serological detection method for humans and different animals.1.Development and identification of monoclonal antibodies against SARS-CoV-2 N proteinThe full-length gene sequence（Gene ID:43740575）of the SARS-CoV-2 N protein in NCBI was used as a template for PCR amplification of the N gene.The target gene fragments were linked to the prokaryotic expression vector pET-28a,pGEX-6P-1 and eukaryotic expression vector pcDNA3.4,respectively.The corresponding recombinant expression plasmids were obtained after PCR verification and sequencing identification.Two prokaryotic expression plasmids of pET-28a-Nand pGEX-6P-1-N were transformed into BL21（DE3）to obtain recombinant expression strains BL21（DE3）-pET-28a-N and BL21（DE3）-pGEX-6P-1N.The recombinant His-N protein with molecular weight of 53 kDa was obtained from strain of BL21（DE3）-pET-28a-N after induced by IPTG and purified by Ni²⁺ column affinity chromatography,while GST-N protein with molecular weight of 73 kDa was obtained from strain of BL21（DE3）-pGEX-6P-1-N after induced by IPTG and purified by GST chromatography.The eukaryotic expression plasmid pcDNA3.4-N was transfected into CHO cells,and the recombinant CHO-N protein expressed in the culture supernatant was purified by Ni²⁺ column affinity chromatography.Western blot results showed that all three recombinant N proteins could specific react with mice or human positive sera displaying a single band with correct molecular weight.These proteins provide good biomaterials for the preparation and screening of monoclonal antibodies.After three times immunizations to mice,mAbs specific recognize recombinant N protein were prepared by classical B-cell hybridoma technology.A total of 12 hybridoma cells that can stably and continuously secrete SARS-CoV-2 N protein mAbs were obtained,named 1E8,4C2,11F4,5D3,8B8,6C5,5E5,5D9,1A2,1C5,1D9,5H9 with cell supernatant titer higher than 1.0×105.The subclass identification results showed that 1 E8,4C2,11F4,5D3,8B8,1A2,1C5,1D9,and 5E5 belonged to IgG1,while 5D9,5H9 and 6C5 belonged to IgG2a.Western blot results showed that all 12 mAbs reacted only with recombinant N protein but not that of empty vectors.The purified 4C2,11F4 and 1A2 mAbs showed high affinity with affinity constant of 1.42×10¹⁰ M^-1,4.4×10⁸ M^-1 and 1.17×10⁹ M^-1,respectively.The results of indirect immunofluorescence test and indirect ELISA based on CHO-N protein showed that all 12 mAbs could specifically bind to SARS-CoV-2 N protein expressed in eukaryotic system,among which 6 mAb of 4C2,8B8,11F4,1A2,1D9 and 1C5 reacted to the SARS-CoV-2 N protein strongly.Furthermore,the recognition ability of the 6 mAbs with N protein of different coronavirus was analyzed by indirect ELISA assay,and the results showed that all of them could react with SARS-CoV-2 recombinant N proteins specifically,indicating the potential for SARS-CoV-2 detection.Based on the relatively stronger specificity and affinity,mAb of 4C2 was selected for the following establishment of the competition ELISA method.2.Establishment and preliminary application of a competitive ELISA method based on monoclonal antibody of SARS-CoV-2 N proteinmAb of 4C2 was selected to establish a competitive ELISA method.The optimal coating concentration for GST-N was determined by ELISA checkerboard method to be 1 μg/mL,and the working concentration of HRP-4C2 enzyme-tagged antibody was 1:12000（99.5 ng/mL）.On this basis,the blocking conditions,sample dilution,enzyme-labeled antibody action conditions,color development time and other experimental conditions of the competitive ELISA were optimized by controlling the single variable method.The final detection steps were set up as following:the antigen was diluted to the working concentration with CBS buffer and coated overnight at 4℃ for 16 h,blocking solution（2%skim milk-PBS）was added and held at 37℃ for 3 h,the optimal dilution factor of serum was 1:10,and the action time of enzyme-labeled antibody was 1 h,and finally develop color at 37℃ for 8 min before detect at OD450 nm.The threshold value of the competitive ELISA method was identified as 27.36%based on the detection results of 60 negative and 60 positive mouse sera.The sensitivity,specificity and stability of the established competitive ELISA method were analyzed respectively.The results showed that the detection limit of this method was 0.106 μg/mL.There was no cross-reaction with animal sera against 19 different pathogenic microorganisms including other coronaviruses,indicating that the specificity of the method was good.The coefficient of variation（CV）value of both the inter-batch and intra-batch replicate tests is less than 15%,suggesting that the method had good reproducibility.The 96.15%sensitivity and 94.12%specificity of the competitive ELISA method was calculated by detection of 43 human serum samples which detected by chemiluminescence immunoassay,displaying better sensitivity than the commercial SARS-CoV-2 Nucleocapsid Protein IgG Antibody ELISA kit.The established competitive ELISA method was used to determine 412 human serum samples,and the overall agreement rate with chemiluminescence immunoassay was 98.78%.Serum samples collected from 34 cats,40 dogs,and 52 pigs were tested with negative results,indicating a low risk of virus spillover to other animals.In this study,a competitive ELISA method based on mAb specific to SARS-CoV-2 N protein was established and applied preliminarily with good specificity and sensitivity,which provided an important technical basis for the serological detection of SARS-CoV-2 infection in humans and different animals.