| Gyrovirus homsa 1(GyH1)is a small,capsidless,single-stranded,cyclic DNA virus with positive icosahedral particles.GyH1 infection primarily causes aplastic anemia,immunosuppression and persistent lymphocytic inflammatory response in the host.In 2018,GyH1 was isolated in our laboratory from broiler chickens with transmissible viral proventriculitis(TVP).In animal regression tests,GyH1 was confirmed to be the causative factor for the development of TVP.TVP can cause anemia and immunosuppression in chickens,leading to severe stunting and growth retardation,which has a significant impact on the performance of broiler chickens and brings huge economic losses to the farming industry.This study was based on colloidal gold immunolayer.In this study,based on the principle of colloidal gold immunochromatography,the reaction conditions were optimized by preparing monoclonal antibodies to GyH1,establishing a colloidal gold test strip assay for GyH1,conducting a comparative analysis of the performance of the test strip,and evaluating the applicability of the test strip in combination with clinical sample testing for monitoring GyH1 infection.VP1 is the major capsid protein of GyH1 and plays a key role in virus assembly,replication and infection.We constructed the VP1 prokaryotic expression vector and expressed the VP1 protein,and verified the purification effect and immunogenicity of the purified VP1 protein by SDS-PAGE and Western blot.In this study,45-day-old New Zealand rabbits were immunized with purified VP1 protein fully emulsified with Freund’s adjuvant,injected subcutaneously at several sites on the back,and the serum was collected after three immunizations,and the VP1 polyclonal antibody was verified by SDS-PAGE and WB.The purified VP1 protein was fully emulsified with Freund’s adjuvant to immunize 6-week-old female BALB/c mice.Myeloma cells(SP/20)were fused with splenocytes from immunized mice and screened,and ascites was prepared by intraperitoneal inoculation of 6-to 8-week-old female BABL/c mice using the obtained hybridoma cells.The ascites was collected and purified to detect the antibody specificity by SDS-PAGE and WB.Colloidal gold solution was prepared by reduction of trisodium chlorogold solution with trisodium citrate to optimize its reaction conditions,and gold-labeled antibodies were prepared using colloidal gold particles of certain size with m Ab labeling.p Ab was wrapped in the detection line and goat anti-mouse Ig G secondary antibody was wrapped in the quality control line,and when the sample to be tested was added dropwise to the sample pad,the gold-labeled antibodies on the binding pad were specifically bound by the siphoning effect of dissolution,and were trapped on the aggregated detection band,which could be The results can be observed by the naked eye.The GyH1 colloidal gold test strips were developed according to the preparation method of colloidal gold test strips.The specificity of the colloidal gold test strips was tested with known viral solutions of GyH1,CAV,ALV-J,REV,Cast V,PCV and Du CV;the sensitivity of the test strips was tested with different concentrations of GyH1 viral solution;the stability of the test strips were stored at 4 ℃,25 ℃ and 37 ℃ for 6 months,respectively,and the stability of the test strips were tested with different samples from the same batch and The performance of the test strips was evaluated by using different samples from the same batch and different batches of the same sample for reproducibility testing.The results showed that the size of VP1 protein was 57 KD,the monoclonal antibody could bind to VP1 protein effectively,and the antigenicity of the mouse-derived monoclonal antibody was high;the particle size of the colloidal gold solution prepared by trisodium citrate reduction was uniform,and the m Ab and p Ab were used as the gold standard antibody and the detection antibody,respectively.The optimal p H of the gold standard complex was 8.8,and the optimal working concentration of the monoclonal antibody was 7.2 μg/m L.The test strip specifically recognized GyH1,and the minimum detection limit of GyH1 test strip was103 copies/μL,which could be stored in a sealed environment at room temperature for 6months.The developed GyH1 colloidal gold immunochromatographic test strips have the advantages of specificity and sensitivity,rapid detection,low cost,and no need for special equipment.To evaluate the GyH1 colloidal gold immunochromatographic test strips,cloacal swabs and serum were collected from a farm in Shandong,and the positive rate reached 7.9%.The results suggest that cloacal cotton swabs may be more suitable for the detection of GyH1 antigen.In this study,we constructed a VP1 prokaryotic expression vector and expressed VP1 protein efficiently,and prepared polyclonal antibodies and monoclonal antibodies from VP1 protein.Based on m Ab and p Ab,we developed GyH1 colloidal gold immunochromatographic test strips and evaluated the performance of the test strips,which proved that GyH1 colloidal gold immunochromatographic test strips have high specificity,sensitivity and stability and can be It can be used for the surveillance of GyH1 infection in primary clinics and provide technical support for the immediate detection of GyH1. |
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