Establishment Of Indirect ELISA And Colloidal Gold Test Strips For The Diagnosis Of Fasciola Hepatica MRLC Antibody

Fasciola hepatica,a parasitic disease caused by Fasciola hepatica,can lead to death in severely infected animals.The disease is widely prevalent worldwide and is classified as one of the important zoonotic diseases by the World Health Organization(WHO).The early diagnosis of liver fluke is particularly important as it causes huge losses to the global livestock sector and also poses a serious health risk to humans.In the present study,the myosin-regulated light chain(MRLC)protein with early diagnostic potential was selected by the group’s proteomic analysis of different stages of liver fluke,and specific primers were designed to amplify the fragment and successfully construct PET-32 aFhMRLC expression vector.The recombinant FhMRLC protein had good immunogenicity by Western blotting analysis after optimizing the induction expression conditions and purifying the protein.The purified recombinant FhMRLC protein was used to establish an indirect ELISA method,and the results showed that the recombinant FhMRLC protein encapsulation concentration was 1.5ng/μL;the optimal dilution of negative and positive sera of sheep liver slice was 1:400;the incubation time of primary antibody was 4 ℃ for overnight encapsulation;the most suitable screening solution was 1% BSA;the incubation time of secondary antibody was 45 min;the color development time was 15 The earliest positive sera from sheep were detected on day 14,and there was no cross-reactivity with Schistosoma haematobium,Schistosoma haematobium,Schistosoma japonicum and Schistosoma bivalve;the coefficient of variation of inter-and intra-plate experiments did not exceed 10%;ELISA was performed on the positive sera of sheep with Schistosoma haematobium that had been verified by the egg test,and all 10 sera were positive,with a 100%compliance rate.The detection rate was 17.42%(62/356)in 356 clinical samples from some areas of Heilongjiang Province.The results showed that the optimum p H of colloidal gold was 8,the optimum protein concentration was 18.75 μg/mL,the concentration of recombinant protein in the detection line was1 mg/mL,and the concentration of rabbit multiple antibody in the quality control line was 1 mg/mL.The first positive sera from artificially infected sheep were detected at day 14.The detection rate of356 clinical samples tested by the established indirect ELISA method was 19.38%(69/356),and the compliance rate with the results of the indirect ELISA method was 89.86%.This method can be used for the early diagnosis of liver flukes.