| Brucellosis is a zoonotic infection caused by Brucella spp.Dogs can be infected with the smooth type of Brucella such as Brucella melitensis and Brucella abortus as well as the rough type of Brucella canis.Dogs infected with Brucella can develop reproductive disorders such as miscarriage and orchitis.A longer period of bacteraemia also occurs,with a high risk of transmission.People may also be infected through direct contact with aborted fetuses and placentas of infected dogs.If a dog is infected with Brucella smootha,it can be detected by indirect ELISA and other serological methods for detecting Brucella smootha.In the case of Brucella canis infection,the presence of antigen self-agglutination in canine sera in agglutination-type tests often results in false-positive tests.Currently,there is no ELISA test for Brucella canis.Therefore,the establishment of scientific and accurate diagnostic methods is the prerequisite and key to the effective prevention and control of Brucella canis infection in dogs.The competitive ELISA antibody test for canine brucellosis uses monoclonal antibodies specific for Brucella canis,making it possible to diagnose canine brucellosis in a variety of animals with good sensitivity and specificity.In this study,monoclonal antibodies against Brucella canis were obtained using Brucella canis RM6/66 as the immunogen in immunized mice after four subclonations.The antigenic epitopes recognized by the monoclonal antibodies were identified by both phage display techniques as well as immunoproteomics.A competitive ELISA method was established with the obtained monoclonal antibodies,reaction conditions were optimized,and the specificity and sensitivity of the method were validated,while clinical samples were tested.The following study results were obtained:1.Four hybridoma cells 1B2,1C5,1D2 and 2E6 secreting monoclonal antibodies against Brucella canis were successfully screened and obtained.The monoclonal antibodies secreted by the four hybridoma cells were all of Ig G2 b subtype and had good stability of secretion.Among them,monoclonal antibody 1C5 reacted specifically with Brucella canis only,and did not react with prevalent strains of Brucella abortus,prevalent strains of Brucella melitensis,Brucella abortus A19 strain and Brucella suis S2 strain.2.After four rounds of selection by phage display technology,the mock linear epitope of monoclonal antibody 1C5 was obtained as "R-D-FGE";the protein was separated by bi-directional electrophoresis and analyzed by protein profiling,and the possible protein targeted by monoclonal antibody 1C5 was initially identified as OMP31,which was expressed in prokaryotic form.Its reactivity with the monoclonal antibody was further verified,and the results showed a positive reaction.3.A competitive ELISA assay for Brucella canis was successfully established using monoclonal antibody 1C5 as a competitive antibody.The antigen concentration was determined to be 1 ?g/m L,and the coating condition was 4 ℃ for 12 h.The closure solution was 5% BSA,and the condition was 37 ℃for 2 h.The dilution of the monoclonal antibody was 1:12000,and the dilution of the serum to be tested was 1:50,and the reaction condition was 37 ℃ for 30 min.The threshold value of blocking rate was determined to be 26.07%.The sensitivity and specificity of the method were further verified,and the results showed that there was no cross-reactivity between the method and Yersinia pestis O9 positive serum,E.coli O157 positive serum and smooth Brucella positive serum;when the Brucella canis positive control serum was diluted to 1:1200,positive results could still be detected;the method and Brucella canis micro-serum agglutination The compliance rate was 99.30%.In this study,we obtained a monoclonal antibody that reacted specifically with Brucella canis only,identified the antigenic epitopes targeted by the monoclonal antibody,and established a competitive ELISA antibody assay for Brucella canis,which is expected to provide a sensitive and specific assay for the diagnosis of Brucella canis. |
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