Development Of Time-resolved Fluoroimmunoassay For Detection Of PCV3 Antibody

Porcine circovirus type 3（PCV3）can infect pigs of different ages and is associated with diseases,such as Porcine dermatitis and nephropathy syndrome,porcine respiratory disease complex,reproductive failure,and multi-system inflammation.Clinically,the prevalence of PCV3 infection increases annually.At present,there is no commercially available vaccine for PCV3.Prevention and control of PCV3 infection mainly rely on accurate detection methods and biosecurity precautions.The serological detection method has the advantages of convenience,rapidity,simple operation,detection of recessive infection,and monitoring antibody level in animals,which is an important reference for the design of disease surveillance and feed management programs on farms.The immunochromatographic assay s a broad market potential,because it has the advantages of efficiency,economy,and applicability to people without professional background knowledge.This dissertation proposes to establish a rapid and accurate immunochromatographic assay for the detection of PCV3 antibody titers in pigs.For this purpose,the following research was carried out.1.Preparation of PCV3 monoclonal antibodyThe nucleotide sequence of the PCV3 ORF2 gene at position 115-642 was used as the target fragment,of which the codon usage was optimized according to the best codon of E.coli for the expression of PCV3 Cap recombinant protein.Western Blot results showed that the recombinant protein had well reactogenicity with PCV3 positive serum.The recombinant protein was used to immunize BALB/c mouse,and two hybridoma cell lines were obtained after several subcloning screenings of hybridoma cells using the cell fusion technique in vitro.Two hybridoma cell lines were named 4B1 and4B3,and secreted the monoclonal antibody（m Ab）subclasses IgG3 and IgG1,respectively.Based on the pCAGGS vector expression system,an indirect immunofluorescence assay（IFA）was carried out to identify the specificity of two monoclonal antibodies.The results showed that both 4B1 and 4B3 reacted specifically with the PCV3 Cap protein expressed in PK-15 cells.2.Development of a Time-resolved fluoroimmunoassay（TRFIA）for PCV3 antibody detectionPCV3 antibody TRFIA was developed based on a competition assay with time-resolved immunofluorescent microspheres conjugated to PCV3 m Ab as the tracer,the PCV3 Cap recombinant protein as the test line of the TRFIA,and the goat anti-mouse polyclonal antibody as the control line of the TRFIA.Results of the experimental analysis indicated that 100μL fluorescent microspheres optimally conjugated with 100μg of 4B1monoclonal antibody,and the optimal encapsulation concentrations of PCV3 Cap recombinant protein and Goat anti-mouse polyclonal antibody were 0.5μg/cm,and 0.3μg/cm,respectively.The best detection time was the 13th min after spotting.The curvilinearity of the TRFIA for detecting PCV3 antibodies was satisfactory,with a curvilinear equation of y=0.4402e^-0.002X,R²=0.9811.The sensitivity was very excellent,with a minimum detection limit of 17.57 mg/L for Igantibodiesdy.The TRFIA exhibited no cross-reaction with porcine circovirus type 2 positive serum,porcine reproductive and respiratory syndrome positive serum,porcine epidemic diarrhea positive serum,and African swine fever positive serum,with powerful specificity.The intra-and inter-batch coefficients of variation of the TRFIA ranged from 1.22%～7.48%,which was less than 10%with a reliable reproducibility.The TRFIA and IFA were simultaneously used for the detection of 60 clinical serums,and the coincidence rate was 88.33%.The results of 585clinical serums detected by the TRFIA showed that the positive rate of PCV3 antibodies was 40.51%,suggesting the prevalence of PCV3 in pigs.In summary,the TRFIA for detection PCV3 antibody has the advantages of convenient operation,rapid detection,high sensitivity,strong specificity,and quantitative detection,which can be used for rapid serological detection of PCV3.