The Screening Of HEV ORF2 Monoclonal Antibodies And Its Application In Virus Detection

Hepatitis E is an infectious disease caused by hepatitis E virus(HEV).The incidence rate of hepatitis E has exceeded that of hepatitis A,making it the main type of acute hepatitis in China.The full-length sequence of the HEV genome contains three open reading frames(ORF1 to 3),of which the ORF2 gene encodes the main structural protein of HEV.The ORF2 protein is highly immunogenic and contains neutralizing epitope-concentrating regions.Currently,HEV antigen detection kit has been developed based on the HEV ORF2 protein and used in clinical diagnosis and treatment monitoring;however,rural areas and urban community grassroot medical units are in need of faster and more convenient HEV antigen detection methods.Previous studies have prepared a number of monoclonal antibodies against HEV ORF2.In this study,the types and affinities,the spatial position characteristics of the monoclonal antibodies were analyzed using indirect ELISA(Enzyme-linked immunosorbent assay)and Western blot(WB)respectively,and the spatial position relationship of monoclonal antibody epitopes was analzed using an competitive inhibition ELISA.On this basis,the dominant epitopes of antibodies were analyzed using vaccinated rabbit serum and serum of naturally infected persons,which provided a theoretical basis for the research of HEV diagnosis,vaccine and infection mechanism.The dominant antibodies were combined,and the double-antibody sandwich method was used to develop immunofluorescent reagents for HEV urine antigen detection,and the sensitivity and specificity of this rapid detection reagent were evaluated.Among the 26 monoclonal antibodies screened,8 monoclonal antibodies with high affinity and broad spectrum for HEV ORF2 antigen were selected.These monoclonal antibodies have a heavy chain type of Ig G and a light chain type of Kappa.Three of these monoclonal antibodies have linear epitopes,and the other five monoclonal antibodies require further analysis.The 8 monoclonal antibodies selected and 3 neutralizing monoclonal antibodies that have been publicly reported are divided into four groups according to the antigenic sites' spatial position relationship.Analysis of dominant epitopes of the antibodies with vaccinated rabbit serum and natural infected serum,the third group of monoclonal antibodies(8 #,15 #,17 #,34 #,58 #,77 #)were found to be dominant antibodies in the serum after vaccination and the serum after natural infection.Among them,17# monoclonal antibody has more obvious blocking effect on antigen and naturally infected serum binding.The dominant monoclonal antibodies were combined in pairs,and the monoclonal antibodies numbered 15 # and 34 # were made into immunofluorescent detection reagent.This reagent can successfully detect the HEV antigen in urine samples with of 76.9%.sensitivity and 96.9% specificity.After optimizing the immunofluorescence rapid detection reagent,the sensitivity and specificity have been improved.After dilution of HEV-positive urine samples in series,the endpoint dilution that can be detected by the immunofluorescence rapid detection reagent is lower than that of the HEV antigen detection kit,which needs to be further optimized.In summary,this study systematically analyzed the affinity,type and epitope of 8 HEV ORF2 m Abs,and found 6 strains of advantageous antibodies in hepatitis E vaccinated rabbit serum and serum of naturally infected HEV patients.One of the monoclonal antibodies had a significant epitope advantage in the serum of naturally infected persons than the vaccinated serum,suggesting that the antibodies produced after vaccine immunization may not be sufficient to fully protect the body from HEV virus infection.The immunofluorescence rapid detection reagent prepared by the dominant monoclonal antibody can detect hepatitis E antigen in the urine of HEV patients,but its sensitivity needs to be further improved.This method is simple and time-saving,and has initially demonstrated potential for clinical application.