ELISA Detection Of Anti-HBV Antibody Based On The Peptide Combination Of Key Epitopes

Hepatitis B(HB),caused by Hepatitis B Virus(HBV)infection,remains one major contagious disease in the world.Previous studies have shown that the PreS1 region of HBV surface antigen plays an important role in HBV infection and life cycle.Hence the detection of anti-PreS1 antibody has attracted more attention recently.Enzyme-linked Immunosorbent Assay(ELISA)is still the most widely used method for antibody detection.Yet,more accurate and effective antibody detection based on ELISA method is still in great need for clinical application.Herein,it is envisioned that the full-length PreS1 protein coating can be mimicked and replaced by the combined coating of key epitopes,which would afford the foundation for the imitation of more complexed sequence pool within the patient group.First,a novel genotyping method based on amino acid sequence of key epitotypes has been developed.Then,an ELISA detection method for anti-PreS1 antibody against the 94-117 epitope(P94 epitope)has been established and optimized,especially in the concentration of coating peptides.Subsequently,genotypy-specific antibodies against P94 epitope were generated by immunizing mice with B and C genotype peptides from P94 epitope,which would be the standard for the quantification of P94 antibodies in patient sera.Next,the level of P94 antibodies has been determined for a large number of patient sera with the optimized ELISA detection protocol and a statistical analysis has been carried out to investigate the correlation between P94 antibody level and HBV DNA copy number and disease stage.Finally,the consistency between peptide-combination detection and full-length detection has been verified.The main results are as follows:1.HBV genotyping based on amino acid sequence of key epitopes.In this work,genotyping of 232 patients with hepatitis B virus was carried out,and an amino acid sequence-based genotyping method was established,which provided a theoretical basis for subsequent studies on the detection of key epitope peptides combined with ELISA antibodies.2.Optimization of ELISA method for P94 antibody detection.The optimal working condition was determined as follows: P94 peptides of genotype B and C were combined for coating and the coating concentration of each was 5 ?g/mL.It is found that the coefficient of variation(CV)of peptide-combination detection is lower than the CV of single-peptide detection,indicating that the ELISA method coated with peptide-combination has higher reproducibility and accuracy.3.Preparation of monoclonal antibodies for the quantification of P94 antibodies.Herein,monoclonal antibodies specific for genotype B or C of P94 epitope(B19 and C04),as well as monoclonal antibody with equal cross-reactivity over B and C genotypes(B11),have been generated as the positive controls for quantitative P94 antibody detection.The cutoff values for positive P94 antibody detection has been decided.4.Statistical analysis of correlation among P94 antibody level,HBV DNA copy number and disease stage.The P94 antibody level in 631 patient samples has been determined and ithe correlation with HBV DNA copy number and disease stage has been investigated.It shows that P94 antibody level within positive range increases with HBV DNA copy number,while decreasing with pregression of disease stage.5.Establishment of ELISA method via the key-epitope peptide combination.PreS1 antibody in patient sera were determined by ELISA methods with various coating antigens,including full-length PreS1 protein,P21 peptide,P94 peptide and combined peptides of P21 and P94 epitopes.Statistical analysis reveals that full-length detection for anti-PreS1 antibody could be replaced by the peptide-combination detection.