Establishment of a competitive ELISA method for detecting antibodies against lumpy skin disease virus
In order to establish an efficient serological detection method for bovine lumpy skin dis-ease virus(LSDV),the prokaryotic expression plasmid pET-25b-RPO30 was constructed after the optimization of the protein codon of RNA polymerase 30 subunit(RPO30)of LSDV.The recombi-nant RPO30 was expressed and purified using anion purification method,which was recognized by the positive serum of bovine LSDV in a Western blot assay,and has good reactivity.The purified RPO30 protein was used to immunize the BALB/c mice.The splenocytes from the immunized mouse were fused with myeloma cells SP2/0.After screening and subcloning,a hybridoma cell line 1H1 with stable antibody secretion was obtained.A competitive ELISA(cELISA)antibody detec-tion method was established by optimizing reaction conditions using recombinant RPO30 protein as the coating antigen.The optimal conditions for the cELISA method were established as follows:using 1 mg/L rRPO30 as the coating antigen in a 100 μL volume overnight at 4 ℃;blocking with 1％BSA at 37 ℃ for 2 h;the optimal dilution of serum to be tested was 1∶2;the optimal dilution of the 1H1 antibody was 1∶2 000;and that of rabbit anti-mouse HRP-IgG was 1∶4 000;the opti-mal color development condition was to react at 37 ℃ for 15 min.In addition,when the cutoff value was set to 0.55,the consistency between the values of sensitivity and specificity and the identifica-tion of serum samples were the largest.Therefore,the cut-off value of PI was set to 0.55.There-fore,samples with 1-S/P＜0.55 were considered negative,and samples with 1-S/P≥0.55 were considered positive.The cross-reactivity with other serum samples including bovine infectious rhi-nobronchitis virus(IBRV),Mycoplasma bovis,bovine viral diarrhea virus(BVDV),bovine brucel-la,and foot-and-mouth disease virus(FMDV)were negative by cELISA.A total of 200 clinical ser-um samples with immune background was confirmed using cELISA,the results showed that it can be used to evaluate clinical vaccine immunity.It is the first to establish cELISA method based on recombinant RPO30 protein,which is a simple,specific,sensitive,and reproducible serological di-agnostic method,providing reliable technical support for the detection and prevention of LSDV.
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