Study On The Immunoassay Assay Based On African Swine Fever DUTPase Nanobodies

African swine fever（ASF）is an acute,hot and highly contagious disease caused by the pathogen African swine fever virus（ASFV）,which has posed a serious threat to the global economy and food security.Due to the lack of effective vaccines and antiviral drugs,it is of great significance to establish a rapid,accurate and applicable immune detection method for ASFV for the prevention and control of the epidemic.Numerous studies have shown that dUTPase（E165R）is an important DNA repair enzyme encoded by ASFV.It can regulate the level of dUTP/d TTP in cells and maintain the fidelity and smoothness of the ASFV genome in macrophages,making it a potential important target for the treatment and detection of ASFV.However,a highly sensitive and accurate immunoassay assay targeting ASFV dUTPase has not been reported.In this project,we constructed point-mutant phage libraries and selected the candidates targeting ASFV-dUTPase by panning.Combined with indirect ELISA,competitive ELISA,double nanobody sandwich ELISA,protein crystallography and isothermal microtitration and so on,the immunodetection assay based on ASFV-dUTPase nanobody was studied.The results of the study are as follows:1.Construction of Nanobody Point Mutation Library:The heavy chain variable region（VH）was randomly mutated by error prone PCR,using the original antibodies scaffolds 1F1,1H4,or 2A12.Three nanobody point mutation libraries were constructed with a capacity of 7.5×10⁷,5×10⁷,5×10⁸.2.Panning and affinity test of nanobody:Three nanobodies with strong affinity,such as 1F1-1F,1H4-11D,2A12-4B,were selected after 4～8 rounds progressive panning with ASFV-dUTPase as antigen.The affinity between Nanobodies and antigen was determined by indirect ELISA.The results showed that the binding constants EC₅₀ of the three nanobodies against antigen were 19.93 n M,52.24 n M and10.18 n M,respectively.3.Study on functional epitopes of nanobody:The nanobody labeled with FLAG-tag and the unlabeled nanobody were paired by competitive ELISA assay as the detection antibody and the capture antibody,respectively.The competitive ELISA assay showed that the epitopes of 1F1-1F,1H4-11D and 2A12-4B did not overlap.Combining protein crystallography and molecular docking to study the intraction between antigen and nanobody,some antigen/nanobody protein crystals had been observed,but they needed to be further optimized for bigger size.The results of molecular docking show that the three nanobodies interact with different amino acid residues on dUTPase,which means that the epitopes interaction with the nanobodies are different.4.Screening of the best nanobody combination:ASFV-dUTPase was determined with the combination of 1F1-1F,2A12-4B and 1H4-11D as the capture antibody and the detection antibody respectively.The data of double antibody sandwich ELISA and ITC showed that 2A12-4B and 1H4-11D-FLAG-tag were the best combination.At the same time,antigen detection by 2A12-4B and 1H4-11D-FLAG-tag sandwich ELISA assay revealed a linear relationship between OD₄₀₅ absorbance and ASFV-dUTPase concentration in the range of 8.55 to 68.44 n M.Therefore,in the subsequent practical application,the antibody combination can be used not only for the qualitative detection of ASFV,but also for the quantitative detection of ASFV.5.Specificity determination of 2A12-4B and 1H4-11D-FLAG-tag antibody combination:ASFV-dUTPase as a positive control,1%BSA and PBS as negative control.Pseudorabies virus,Classic African swine fever,Porcine circovirus type 2,Porcine reproductive and respiratory synthesis virus vaccines samples were used to test the specificity of the nanobody combination.The data of the double antibody sandwich ELISA assay showed that the antibody combination had great specificity and sensitivity.6.Prokaryotic expression of gold-labeled nanobody:the murine m Fc fragment was coupled with the 2A12-4B heavy chain variable region via homologous recombination to form recombinant vector,and BL21（DE3）strain was used for protein expression.The detection of SDS-PAGE and WB showed that the recombinant proteins could be successfully expressed in prokaryotic cells and had good reactivity,which laid the foundation for the rapid and specific detection of the immunocolloidal gold chromatograph assay.In conclusion,based on the anti-ASFV-dUTPase nanobodies preserved in the laboratory,this project further optimized to enhance the specific binding and weaken the nonspecific binding.To facilitate the development of the immunological assay,the best antibody combination was established,and the double antibody sandwich ELISA technology was established.At the same time,based on the qualitative analysis of the antigenic epitopes of these three antibodies,the amino acid identification was carried out,which laid the foundation for ASFV-dUTPase as a target for diagnosis and treatment.