Systematic Analysis Of HCV Core Protein Antigenic Epitopes And Establishment Of Highly Sensitive Immunoasassys With Controlled Signal Amplification System

Backgrounds and objectivesHepatitis C virus(HCV)infects 200 millions of peoples worldwide,and is highly prevalent in China as well.Among HCV infected individuals,30%of them resolved spontaneously while 70%of them became chronic infection and 10%-20%of chronic carriers furtherly developed to liver cirrhosis,liver failure or primary hepatocellular carcinoma(HCC).Normally HCV infection is diagnosed or detected by ELISA for antibody,PCR for viral RNA or by both.Currently,among individuals with "antibody positive" to HCV by a single ELISA,approximately 1/3 are false positive,1/3 are antibody positive but RNA negative(HCV Ab+/RNA-),and 1/3 are both positive for antibody and RNA(HCV Ab+/RNA+).Besides detection of HCV antibody and RNA,as the HCV core protein antigen(HCVcAg)is in presence of whole HCV replication course,which is considered as an important marker for diagnosis of chronic HCV infection in clinical practice.At current commercial market,only few diagnosis kits are available for HCVcAg,including two international assays from Ortho and Abbott,and two domestic assays from Hunan Runkun and Shangdong Laibo,of which all are in lower sensitivity and coverage for detection of HCV infection.Hence detection of HCVcAg is still a global technical obstacle,in which the major difficulty is not well realized for HCVcAg antigenic epitopes and lacking of high affinity monoclonal antibodies(mAbs)and highly sensitive assays.In this thesis,regarding to the lack of critical reagents and the weakness of assays for HCVcAg detection,the study is to analyze the epitopes of HCVcAg fully and systematically,to produce a panel of applicable monoclonal antibodies and to develop highly sensitive immunoassays for specific detection of HCVcAg in blood donation screening and clinical diagnosis.MethodsThe antigenic epitopes within HCVcAg were explored systematically by using syntheitic peptides,in which the conserved peptides were used for immunizing BALB/c mice for examining peptides' antigenicity.A panel of monoclonal antibodies(mAbs)recognizing HCVcAg were generated by fusing immunized spleen cells with murine myeloma.HCVcAg epitopes were carefully identified by the selected mAbs,which recognized the multiple epitopes of natural HCVcAg.The highly sensitive controlled signal amplification system(SAS)with gold nanoparticles(AuNPs)or fluorescent lanthanide Eu+3 nanoparticles(EuNPs)was designed.By using the paired mAbs and the controlled SAS,the highly sensitive lateral flow immunoassay(LFIA)or ELISA were developed for detecting HCVcAg in blood donation and clinical diagnosis.Results1.Antigenic epitopes of HCVcAgFive conserved peptides 21-40aa,41-60aa,51-70aa?101-120aa and 121-140aa derived from HCVcAg were selected by analyzing amino acid sequence of HCV core protein.By using these peptides for immunizing BALB/c mice,57 reactive mAbs were selected,which specifically recognized natural HCVcAg.Among these 57 mAbs,46 mAbs reacted with the 41-60aa,100-120aa and 121-140aa epitopic peptides,which revealed these three new antigenic epitopes of HCVcAg in the world.In this study,21-40aa?41-60aa?101-120aa peptides could induce high affinity antibodies and modified the description of HCV core protein having only a 21-40aa epitope.This novel recognition regarding to the HCVcAg epitopes provides the theoretic support and key reagents for developing immunoassays for detecting HCVcAg by sandwich antibodies.2.Highly sensitive controlled signal amplification systemIn contrast to conventional signal amplification systems in which signal was amplified fully without controlling and possibly produced non-specific reaction,a controlled signal amplification system(SAS)with two kinds of formats was developed for use in highly sensitive immunoassays as described in the below(1)A controlled-SAS for LFIAThis format of controlled signal amplification system comprises of nanoparticles(AuNPs or EuNPs)labeled detection antibody to target antigen,biotinylated capture antibody to target antigen,and coated antibody to biotin.When the target antigens are presence in sample,they are bound by the biotinylated capture antibody and the nanoparticle labeled detection antibody to form the dendritic complexes,which are extracted by the coated antibody to biotin,and then furtherly are detected by the highly sensitive Controlled-SAS basing on LFIA Signal intensity is massively amplified with this SAS,which is positively correlated with the concentration of target antigen in blood sample and was assessed by eyes or strip scanner.The Controlled-SAS LFIA works only when target antigen presents in the sample.This system was demonstrated nine folds higher than conventional assay,which massively improved its sensitivity and specificity for detecting for HBeAg from plasma samples and could be applied in establishing highly sensitive immunoassays for HCVcAg detection(2)A controlled-SAS for ELISAThis format of controlled signal amplification system comprises of nanoparticles(AuNPs)conjugated with bi-molecules of anti-biotin antibody and horseradish peroxidase(HRP),biotinylated detection antibody to target antigen,and coated capture antibody to target antigen.When target antigens are presence in sample,they bind to the coated capture antibody and biotinylated detection antibody,furthermore bind to bi-molecules of anti-biotin antibody and HRP conjugated on nanoparticles.HRP in those nanoparticle complexes indirectly detects the presence of target antigens by color indicator from substrate reaction.This controlled SAS has resolved the non-specific reaction weakness of biotin-avidin system and largely improved the sensitivity of conventional ELISA.3.Highly sensitive controlled-SAS immunoassays for detection of HCVcAg(1)A controlled-SAS LFIA for detecting HCVcAg.Based on lateral flow immunoassay,a highly sensitive and specific Controlled-SAS LFIA strip was developed for rapidly detecting HCVcAg in blood samples.A LFIA strip comprised of a sample pad,a conjugate pad,nitrocellulose(NC)membrane,and an absorbent pad and polyvinyl chloride(PVC)baseboard.There was anti-biotin antibody was coated on the test(T)line.The clinical samples of plasma or sera were pretreated for denaturing HCVcAg and then were mixed with biotinylated capture antibody and EuNPs labeled detection antibody for capturing of the denatured HCVcAg.The complexes of EuNPs-antibody-HCVcAg were separated by centrifugation,which were resuspened and further detected by the strip.The sensitivity of this LFIA strip for detecting HCVcAg was equal to HCV RNA 105IU/mL in plasma samples by qPCR.By detecting 41 clinical samples with HCV RNA,the Controlled-SAS LFIA and the conventional ELISA were able to detect HCVcAg from 53.7%or 56.1%samples(P>0.5),respectively.This highly sensitive LFIA for detection may provide a simple,rapid and specific test strip in clinical diagnosis of HCV infection(2)A Controlled-SAS ELISA for detecting HCVcAg.In this assay the coated capture antibody and biotinylated detection antibody bind to sample's HCVcAg in sandwich format.These HCVcAg complexes in the plate were detected under substrate reaction by the Controlled-SAS through its conjugates with bi-molecules of anti-biotin antibody and HRP.As normally HCVcAg is in the form of complex with antibody in HCV carrier's blood,the sample processing solution has been optimized for 5.5M urine,5%Tween 80 and 1%Triton X-100.Optimal conditions of antibody concentration and reaction were determined for detecting HCVcAg in plasma samples by the Controlled-SAS ELISA.In comparison with conventional ELISA,the Controlled-SAS ELISA was 10 times more sensitive in detection of HCVcAg,in which HCVcAg positive samples were 91.1%consistent with HCV RNA detection but conventional ELISA was 55.7%consistent with HCV RNA detection(P<0.01).The Controlled-ELISA overcame the technique weakness of current assays for detection of HCVcAg,which could be used in blood donation screening and clinical diagnosis for HCV infection.Conclusions1.The monoclonal antibodies were generated against peptides 21-40aa,41-60aa,101-120aa and 121-140aa derived from HCV core protein and the recognition peptides were identified as new antigenic epitopes of HCVcAg,which were as target sites for detection and provided molecular mechanism support and key reagents in immunoassays2.A novel controlled signal amplification system(SAS)and its modification format with nanoparticle conjugates labeled with bi-molecules of anti-biotin antibody and HRP were established.These two systems can massively enhance the sensitivity retain the specificity of lateral flow immunoassay(LFIA)or ELISA3.A highly sensitive Controlled-SAS LFIA and a highly sensitive Controlled-SAS ELISA with multiple epitopic mAbs were developed for detecting HCVcAg in blood samples from blood donation screening and clinical diagnosisInnovation and significance1.This study systematically analyzed HCVcAg epitopes at first time in the world.Beyond the known epitope 21-40aa,the peptides 41-60aa,101-120aa and 121-140aa of HCV core protein were found able to induce specific antibodies,which were further identified as new antigenic epitopes and targets for detection of HCVcAg in immunoassays2.A panel of high affinity monoclonal antibodies was newly produced by using epitopic peptides 21-40aa,41-60aa and 101-120aa,which would provide the key reagents for developing immunoassays with double antibody sandwich method.3.Two novel controlled signal amplification systems(SAS)were innovated,which would massively enhance the sensitivity of immunoassays for HCVcAg detection.The highly sensitive Controlled-SAS immunoassays might potentially replace the conventional LFIA and ELISA with the double antibody sandwich format.4.The developed highly sensitive immunoassays with Controlled-SAS could provide a reference model for detecting other target antigens such as HBeAg in clinical diagnosis.