Development Of Chemiluminescence Immunoassay Methods For Rapid Detection Of Antibodies Against Foot-and-Mouth Disease Virus Serotype O And 3ABC

Foot-and-mouth disease?FMD?is an acute,highly contagious animal disease,which is caused by Foot-and-mouth disease virus?FMDV?,and has led to serious losses in the farming industry and the trade of animal products.In developing countries,the control and eradication of FMD rely upon vaccination,in which the inactivated vaccine is predominant.A series of purification methods were used to removed non-structual proteins?NSPs?in the preparation of inactivated vaccine,but NSPs antibodies still were detected in cattle that have been repeatedly immunized in some resports,which cause serious interference to distinguish infected from vaccine animals?DIVA?.Therefore,it is necessary to develop a quantitative detection method of residual NSP for the evaluation of the vaccine.Meanwhile,it is also impotant to develop a sensitive and rapid diagnostic method to DIVA.Furthermore,the diagnostic method that can realize the inter-type differentiation is of great significance to evaluate the immune effect of FMD vaccine,guide prevention and control,and establish scientific immunization programs.1.Identification of three linear B cell epitopes against NSP 3ABC of FMDV using monoclonal antibodiesIn this study,the minimal epitopes“⁹²EYIEKA⁹⁷”,“²³EGPYAGPLE³¹”and“²⁰⁹EPHH²¹²”identified by three monoclonal antibodies?MAbs?2G5,9E2 and 1E10 respectively were determine.Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes.The sequence alignment analysis indicated that the epitope identified by 2G5 is relatively conserved,and the epitope indentifies by 9E2 is highly conserved in 3B2 among different FMDV isolates.And the four-amino acid epitope is the first reported to date that is recognized by 1E10.These results provide a theoretical basis and valuable insight into the diagnosis of DIVA and NSP residual evalution in inacticated vaccines using these MAbs.2.Development of a competitive CLIA based on double MAbs for the detection of FMDV NSPs antibodiesThe double MAbs competitive CLIA?3A+3B CLIA?for the detection of FMDV NSPs antibodies was developed which used FMDV 3ABC non-structural protein as antigen and poly-antibody against 3ABC as capture antibody and two MAbs against 3A and 3B respectively as detector antibodies.The cut-off?40%,40%,50%,respectively?,diagnostic sensitivity?98.13%,95.71%,96.15%,respectively?and diagnostic specificity?99.51%,99.43%,98.36%,respectively?were determined by detecting the sera from pigs,cattle and sheep with known background.The method can detecte susceptible animals,and is not restricted by animal species,and greatly reduce reaction time?only need 15 min?and washing times?washing only once?.3.Development of a CLIA to detecte NSP residual in FMDV inactivitied antigen and inactivitied vaccineIn this study,a double antibodies sandwich CLIA was developed by using poly-antibody against 3ABC as capture antibody and 9E2-HRP as detector antibodies to quantitatively detecte the NSP residual in FMDV inactivitied antigen and inactivitied vaccine.And the optimum demulsification methods for the detection were screened.Furthermore,the inactivated antigen and inactivated vaccine mainly contain 3AB,3ABB and 3ABBB,which was detected by WB using9E2-HRP as detector antibodies.4.Development of an indirect CLIA to detect the antibodies against FMDV O in sera from pigsA multi-epitope-based indirect CLIA was developed to detect antibodies against FMDV O in sera from pigs.The cut-off,diagnostic sensitivity and diagnostic specificity were determined by detecting the sera from pigs with known background.PP?3.5%of test samples were considered as positive,PP<2.2%of these were considered as negative,and 2.2%?PP<3.5%of those were considered as doubtful.The method has high sensitivity,good broad spectrum?good reaction with antibodies induced by other isolates within the same serotype?,and good specificity.The immune protection value of M2-CLIA measured inactivated vaccine was determined indirectly by the antibody titer of O-LPBE.And the immune protection value of M2-CLIA measured epitope vaccine was determined by the clinical symptoms of post-immunization challenge.