Establishment And Application Of Indirect ELISA And Colloidal Gold Test Strip Based On FHH Protein For Fascioliasis Diagnosis

Fascioliasis is an important zoonotic parasitic disease caused by Fasciola hepatica（F.hepatica）and Fasciola gigantica（F.gigantica）,which is parasitic in many animals such as cattle,sheep and human bile duct.Among them,the disease caused by F.hepatica not only seriously affects the development of animal husbandry,but also poses a certain threat to human health.At present,there is no efficient vaccine for F.hepatica,so timely diagnosis before the widespread spread of F.hepatica is particularly important.The screening of specific antigen genes for F.hepatica is of great significance for the development of sensitive and specific diagnostic methods related to this disease.Therefore,this study screened specific candidate diagnostic antigens for F.hepatica,conducted bioinformatics analysis and expression identification,established indirect ELISA and colloidal gold strip diagnostic methods for F.hepatica,and tested clinical serum samples to provide specific candidate antigens and diagnostic technical support for the diagnosis of F.hepatica.Screening of F.hepatica-specific antigen genes and expression of recombinant protein FHH:Through screening of specific antigen genes from the c DNA expression library of adult worms of F.hepatica,8 candidate diagnostic antigens of F.hepatica were successfully obtained,including 2 reported diagnostic antigens of F.hepatica,namely cathepsin L and cathepsin B,and 6 newly discovered candidate antigens of F.hepatica,After comparison,the specific antigen of F.hepatica D915＿009456 was selected for follow-up study.First of all,we were analyzed D915＿009456 by bioinformatics and predicted to be a F.hepatica hydrolase（FHH）protein.At the same time,the FHH protein of F.hepatica was successfully purified and identified.Establishment and application of an indirect ELISA method for diagnosis of F.hepatica:An indirect ELISA method for diagnosis of F.hepatica based on FHH recombinant protein was established.The optimal antigen coating amount for this diagnostic method was 0.75μg/hole;The best sample dilution is 1:800;The best sealing agent is 1%BSA;The optimal dilution concentration of the second antibody is 1:6000;The optimal substrate reaction time is 20 minutes;The positive serum sensitivity was 1:3200;There was no cross reaction between the method and the positive sera from sheep infected with Schistosoma ovis,Neosporidium ovis,and Haematococcus spiralis;The coefficient of variation in both intra and inter batch tests was less than 6%.Using this method and the commercial kit,80 sheep serum samples from Inner Mongolia were tested,and the positive rate of both methods was 17.5%（14/80）.Establishment and application of the colloidal gold test strip diagnosis for F.hepatica:A colloidal gold test strip diagnostic method for F.hepatica based on FHH recombinant protein was established.The optimization results show that the best p H value for the preparation of colloidal gold solution adding 2μL 0.2 M K₂CO₃ solution to 1mL solution;The optimal labeling amount of colloidal gold SPA protein is 6μg;The optimal adding concentration of FHH protein for the test line（T line）is 0.5 mg/mL;The optimal adding concentration of rabbit anti-SPA secondary antibody for the control line（C line）is 0.125 mg/mL;The positive serum sensitivity is 1:80;There was no cross reaction between the method and the positive sera from sheep infected with Schistosoma ovis,Neosporidium ovis and Haematococcus twist;The repeatability test results are good;The storage life test showed that the strips could be stored for 90 d,90 d and 60 d at 4℃,room temperature and 37℃,respectively.The colloidal gold strip diagnostic method based on FHH was used to detect 80 sheep serum samples in Inner Mongolia,and compared with the established indirect ELISA method and commercial kit,the results showed that the positive rates of the three methods were all 17.5%（14/80）.To sum up,this study obtained the specific diagnostic antigen FHH of F.hepatica by screening the cDNA expression library of adult worms of F.hepatica,and successfully established an indirect ELISA and colloidal gold strip diagnostic method for F.hepatica based on this antigen.The two established diagnostic methods were used for preliminary detection of clinical samples,and it was evaluated that the two diagnostic methods have good sensitivity and specificity,which has certain clinical application value,It provides a new candidate diagnostic antigen for the diagnosis and control of F.hepatica.