Development Of A Colloidal Gold Strip For Rapid Detection Of Antibody To Toxoplasma Gondii In Canine

Toxoplasmosis is a worldwide popular parasitic zoonoses caused by Toxoplasma gondii, almost can infect all warm-blooded animals and some cold-blooded animals, with a very wide host range. On the one hand, it can cause pregnant women abortion, fetal abnormalities, and congenital defect or stillbirth, causing serious consequences; on the other hand, it can also lead immunocompromised populations such as AIDS patients, cancer patients to myocarditis, pneumonia or fatal encephalitis et al. People get infected by Toxoplasma gondii mainly through intake undercooked meat containing parasite cyst or the fruits, vegetables and water polluted by T. gondii oocyst. Therefore, monitoring and prevention of T gondii infection in animals can effectively control the spread of the parasite. Dogs play an important role in spreading the parasite to human, they can carry oocyst to human living area, moerover, if people eat dog containing with T gondii cyst may get infected. At present, modified agglutination test(MAT), indirect haemagglutination assays(IHA), indirect fluorescent antibody test(IFA), enzyme-linked immunosorbent assays(ELISA), etc, are mainly clinical serological detection methods of toxoplasmosis, with good sensitivity and specificity. But they need to be done under laboratory conditions, and need experienced technologist and related apparatus, equipment, not suitable for testing in the wild environment. While Immunochromatographic Assay as an emerging rapid detection method completely has the potential to undertake the T. gondii detection task. Research target:Express GRA1, GRA7 recombinant protein in laboratory environment. Then establish indirect ELISA method with the proteins for detecting serum specific antibody Ig G against Toxoplasma gondii, comparing with T. gondii soluble antigens(TLA), and choose a more optimal recombinant protein. Produce rabbit polyclonal antibody against the selected protein, and then establish the Immunochromatographic Assay to detect serum specific Ig G antibody against Toxoplasma gondii. Research contents:1 Expressing and purificating T. gondii recombinant protein in laboratory V environment, then establish and evaluate indirect ELISA method to detect T. gondii specific Ig G antibody in the serum of dogs for further research.2 Preparing, purificating and concentrating of the polyclonal antibodies of the choosed ideal protein.3 Using of the optimal recombinant protein produce rabbit polyclonal antibody to establish Immunochromatographic Assay, compare and evalute the results with MAT and IFA. Research method:1 Expression and purification of GRA1 and GRA7 proteinsCulture and collect the DE3 E.coli strains which contain recombinant plasmid p ET28-GRA1 or p ET28-GRA7 by shaker, then extract target protein by not soluble protein extract cracking. Verify the extracted proteins by SDS-PAGE and Western Blot. Dialysis and concentrate the proteins.2 Preparation of T. gondii soluble antigens(TLA)Revive T. gondii saved in liquid nitrogen and inoculate the parasite to Vero cells. Collect and freeze thawing T. gondii tachyzoites, break tachyzoites by hyperacoustic, save the supernatant at-20℃ with a concention of 1mg/m L.3 Evaluation and establishment indirect ELISA for detection of Dog serum T.gondii specific Ig G antibody.Microplates were coated with GRA1, GRA7 or TLA with a concentration of 5μg/m L, 5μg/m L, 10μg/m L, respectively. Then after blocked by skim milk powder over night, washed and added with canine serum, and incubated for 1h at 37°C, then 100 μl of horseradish peroxidase-conjugated sheep anti-dog Ig G antibodies was added. After incubation for 1h at 37℃ and washing, color was developed by the addition of TMB for 20 min, and stopped by 2M H2SO4. The optical densities(ODs) were measured at 450 nm in a microplate reader. Compare the ELISA results with the gold standard method MAT and IFA, analyze the date by SAS software, and verify the feasibility of the ELISA method. Lastly, ELISA test results were analyzed by Receiver Operating Characteristic curve and evalute the sensitivity and specificity of the metheds. Choose a better protein to establish the Immunochromatographic Assay.4 Production of rabbit anti-GRA7 polyclonal antibody1mg of GRA7 protein was mixed with same volume of Freund’s Complete Adjuvant into water-in-oil state. Moreover, the mixture was injected to the rabbit. At 2w, 4w, 6w after the first injection of the protein, GRA7 protein and Freund’s incomplete adjuvant was mixed and injected to the rabbit. 7 days after the last injection, a small amount of blood serum was taken to detect antibody titers and activivity. Ig G antibody was purified With protein A pillars, and dilluted to 1mg/m L after dialysis and concentrate, stored at-20°C until used.5 Establishment and evaluation of Immunochromatographic AssayColloidal gold was prepared by sodium citrate reduction method, after optimize the protein and PH value, the colloidal gold coated with GRA7 protein was sprinkled on the cellulose membrane. Draw GRA7 protein to the test line, and rabbit anti-GRA7 polyclonal antibody to the control line. detecting dog serum with the colloidal gold strip, and compare the results with MAT and IFA. Research results:1 Successfully established indirect ELISA methods in detecting Toxoplasma gondii specific Ig G antibody in the serum of dogs coated with GRA1, GRA7, TLA at a concentration of 5μg/m L,5μg/m L,10μg/m L as antigen, respectively. The diluted concentration of dog serum is 1: 50. The diluted concentration of HRP-labeled rabbit anti-dog antibody is 1: 20000. Name the three indirect ELISAs of GRA1-ELISA, GRA7-ELISA, TLA-ELISA. The positive rate of the ELISAs is 16.2%, 17.0%, 16.2%, respectively. The canine serum samples were detected for anti-T.gondii antibodies by MAT and IFAT, showing that the seroprevalence was 16.2% by TLA- and GRA1-ELISA, and 17.0% by GRA7-ELISA. There was no significant difference between positive and negative results when comparing TLA-, GRA1-, and GRA7- ELISA results with MAT and ELISA using Mc Nemar chi-square(P> 0.05). The Kappa analysis of the ELISAs was Kappa=0.7491,(95%CI:0.6390-0.8592), Kappa=0.8631,(95%CI:0.7803-0.9459), Kappa=0.8327,(95%CI:0.7409-0.9245), respectively. The Kappa values of three motheds is GRA7-ELISA> TLA-ELISA> GRA1-ELISA, showing a better result of GRA7-ELISA.ROC analysis revealed an area under curve(AUC) of 0.957(95%CI, 0.919-0.995) for GRA1-ELISA, 0.973(95%CI, 0.955-0.991) for GRA7- ELISA, and 0.948(95%CI, 0.911-0.986) for TLA-ELISA, respectively. The AUC of GRA7-ELISA is more closer than other motheds, showing that GRA7 protein has a better antigenicity, and can be used in Immunochromatographic Assay.2 Successfully produce rabbit anti-GRA7 polyclonal antibody. The titers of antibody tested by GRA7 based ELISA is higher than 12800, TLA based ELISA is 3200. Tested by Western blot, the antibody have reactiveness with GRA7 and TLA proteins at the specific place. The best protein coating p H value is 8.0, and the best protein coating concentration is 15.6μg/m L. The best concentration of GRA7 protein drawing at T test line is 0.5mg/m L, and the same concentration to the rabbit antiGRA7 polyclonal antibody at the C control line. Stability test demonstrate that the strips were effective at least one month at room temperature, and at least half year at 4℃.Test the canine serum samples by GRA7 based colloidal gold strips, the positive rate of the samples is 15.8%, lower than the positive rate of GRA7-ELISA. There was no significant difference between the different results when compared with MAT and IFA using Mc Nemar chi-square analytical method, while the same samples show a Kappa value of Kappa=0.7889,(95%CI:0.6864-0.8914), indicating a higher consistency of the two motheds. A substantial agreement of 94.2% showing a sensitivity of 79.5% and a specificity of 97.2%. Research conclusion:1 Successfully developed Indirect ELISAs based on GRA1, GRA7, TLA protein, and the results of clinical tests indicating that GRA7-ELISA is better than GRA1- and TLA-ELISA.2 Successfully prepared the rabbit anti GRA7 protein polyclonal antibody. ELISA and Western Blot verified that the polyclonal antibody showed a perfect reactivity with GRA7 and TLA.3 A colloidal gold strip for rapid detection of antibody to Toxoplasma gondii in canine was developed, a sensitivity of 79.5% and a specificity of 97.2% indicating that the strip can be used to detect dog toxoplasmosis as a rapid method.