Preparation Of Cathepsi L1 Monoclonal Antibody Against Fasciala Gigantica And Establishment Of Double Antibody Sandwich ELISA

As a food-borne parasitic parasite Fasciola gigantica causes serious harm to the development of cattle and sheep breeding industry and human health.At present,prevention,control,diagnosis and preventive measures for fascioliasis in China are still incomplete and there is a lack of low-cost commercial kits,especially it is more difficult to diagnose the disease in underdeveloped areas.In recent years,several studies have reported that Cathepsin L1 is highly expressed in Excretory-secretory product(ESP)from F.gigantica and has good potential in immunity and early diagnosis of Fasciolasis.This study intends to obtain pET-28a(+)-rFgCatL1 through the prokaryotic expression plasmid of rFgCatL1 constructed in preliminary work and prepare mouse rFgCatL1 specific monoclonal antibodies and polyclonal antibodies.Based on this,the double antibody sandwich ELISA was constructed to facilitate clinical detection and epidemiological investigation.In this experiment,the pET-28a(+)-rFgCatL1 prokaryotic expression plasmid constructed in the early stage of the laboratory was used to perform rFgCatL1 expression and purification.The purified protein was used to prepare polyclonal antibodies and specificity and sensitivity were identified.BALB/c mice were immunized with rFgCatL1 and isolated mouse spleen cells were fused with SP2/0 cells to construct positive hybridoma cell line.Supernatant antibody titer,subtype and specificity of positive hybridoma cell line were identified and ascitic fluid from mouse was prepared.Using the prepared anti-rFgCatL1 monoclonal antibody and polyclonal antibody,the double antibody sandwich ELISA of Fasciola gigantica was constructed using to facilitate clinical detection and epidemiological investigation.The results showed rFgCatL1 protein was successfully obtained with a molecular weight of 39 ku,which was in line with the expected value.The prepared anti-rFgCatL1 polyclonal antibody has a titer of more than 128 000.Through Western-blot analysis and specific detection,the prepared polyclonal antibody can specifically bind FgESP.A total of 140 hybridoma cells were obtained after selective culture and 8 of them were positive cell lines.5D5 and 7G6 are strong positive strains.After multiple subcloning screens and subcultures,the antibodies secreted in the cell supernatant were stable,with titers of 29 and 210 respectively.Western blot and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was Kappa type,both of which could specifically bind FgESP.The cell lines were used to prepare mouse ascites,with ascites titers of 107 and 108.Comparing the antigen binding sensitivity of the two monoclonal antibodies,7G6 was used as the coating antibody,and anti-rFgCatL1 coupled HRP was used as the enzyme-labeled secondary antibody.Using direct ELISA detection,the HRP-anti-rFgCatL1 polyclonal antibody titer reached 1:64 000.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL,the dilution concentration of Anti-rFgCatL1 polyclonal antibody was 25 μg/mL,the dilution of Don-HRP-Conjugated is 1:4 000,5%skimmed milk powder was selected as the blocking solution and the color development time is 25 min.After verification,it can recognize the minimum antigen concentration of 0.625 μg/mL and did not bind to the front and rear disk antigen,trypanosoma antigen and Toxoplasma antigen.The critical value was 0.221.Using this method,47 parts of buffalo serum and 47 parts of goat serum that were positive by indirect ELISA method were detected,and the positive results were 72.3% and 78.7%.In this study,anti-rFgCatL1 monoclonal antibody and polyclonal antibody were successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed,which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.