Study On The Immunodiagnosis Of Toxoplasmosis And Angiostrongyliasis

Part I Construction and application of the standard ELISA kits based on the recombinant SAG1 of Toxoplasma gondiiInfections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and animals. T.gondii infection are very harmful to human. Diagnosis is very important for the control and prevention of toxoplasmosis. However, some problems and disputes such as diagnostic standard and efficiency still need to be solved. At present, most of the commercial kits use a native soluble antigen prepared from tachyzoites of T. gondii. This kind of crude antigen is too complex to standardize. The use of recombinant antigens would allow better standardization of the tests and reduce the costs of production.The major surface antigen 1 (SAG1) of T. gondii is one of the most potential diagnostic molecules. In our lab, SAG1 has been expressed as a soluble fusion protein in E. coli, and then confirmed to have a good immunoreactivity. The standard ELISA kits were constructed and applied based on the recombinant SAG1 (rSAG1) in this study.1. Identification of the standard seraThe standard sera are crucial for evaluating the quality of diagnostic kits. Toxoplasma-slide HRP-enzyme immunostaining (TSHE) and Immuno-Blotting(IB) were developed based on the reference sera control such as McAb to SAG1, sera of normal rabbits and rabbits vaccinated by tachyzoites' soluble antigen, which wereidentified by immunoelectron microscope. Then, the positive and negative sera were identified by TSHE and IB.2. Expression, purification and identification of rSAGlIt is needed for diagnostic antigens to produce in a large scale in the construction of the standard kits. Three kinds of techniques, ordinary shaking and fermentation and high-density fermentation, are developed to obtain rSAGl. These results indicated that the yield of rSAGl could reach to 1.65g/L, 1.82g/L and 6.74g/L respectively using the three kinds of techniques. Comparing with ordinary fermentation, high-density fermentation was enough to satisfy the request of a batch production of rSAGlwith lower cost.In order to get the best purification technique of rSAGl and explore whether the purity of rSAGl affecting the detection efficiency, three purification methods, Ni-NTA affinity chromatography and Sephadex-G75 chromatography and electroelution from SDS-polyacrylamide gels , are performed and optimized. SDS-PAGE analysis shows that the purity of rSAGl, obtained by one step purification of Ni-NTA chromatography , two steps purification of Ni-NTA and Sephadex-G75 chromatography, Ni-NTA chromatography combined with electroelution, were 72.36%, 98.54% and 97.39% , respectively. The IgG-ELISA with rSAGl purified by two steps purification of Ni-NTA combined with Sephadex-G75 chromatography appeared a better sensibility and specificity.3. Analysis of the effect factors in the rSAGl -IgG-ELISA systemTo analyze the effect factors of rSAGl-IgG-ELISA system and find a good way to solve the problem, six kinds of ELISA projects were established with the different antigens such as the soluble antigen of tachyzoite, purified rSAGl before and after enterokinase cleavage. These results indicated thatfor sensitivity, there was no significant difference among the six ELISA projects. The fusion carrier protein thioredoxin could cause non-specific reaction, which could be controlled by the sera pre-treatment with thioredoxin absorbent in the project. The ELISA project with the high purity rSAGl fusion protein versus the sera pre-treatment showed a higherspecificity in the sera detection. So, the high purity rSAGl fusion protein could be used to instead of native tachyzoite soluble antigen in construction of the standard IgG-rSAGl-ELISA kit of T. gondii.4. Construction of the rSAGl-IgM-ELISA systemThe detection of T. gondii-specific IgM is the most common method in the determination of the recent infection. However, variability and non-specificity really existed among the commercial available kits. The IgM detecting systems rSAGl-ELISA and TOXO-ELISA, using the tachyzoite soluble antigen, were constructed. Through the detection of T. gondii-specific IgM positive and negative sera, the results showed that the sensibility of rSAGl-ELISA was the same as TOXO-ELISA, whereas, the specificity of rSAGl-ELISA is higher than TOXO-ELISA. It suggested that the high purity rSAGl could be used as the diagnostic antigen instead of the native antigen for establishing the standard rSAGl-IgM-ELISA kit of T. gondii.5. Construction and application of the standard rSAGl-ELISA kitsBased on the above studies, the manufacture procedures of the rSAGl-ELISA kits were further optimized as follow: (1) The ELISA plates were coated with the best coating concentration of the high purity rSAGl. (2) The best blocking buffer and sera dilution buffer were chosen from different solutions. (3) The reaction time, washing conditions and the concentration of the second HRP-labeled antibodies were all optimized by chessboard screening methods.According to the results of detecting 1170 cases of human sera, the average value and SD, CV and critical value were all established. Combined with the positive and negative reference sera, stable concentrated washing buffer, substrate buffer, staining buffer and package boxes, the standard rSAGl-IgG- ELISA and rSAGl-IgM-ELIS A kits were successfully constructed.Part II. Screening, identification and application of the diagnostic antigen(s) of Angiostrongylus cantonensis and the construction of a diagnostic system for angiostrongyliasisAngiostrongylus cantonensis was first isolated from a rat by the Chinese parasitologist Dr. Chen in 1935 in Guangzhou, China. This parasite has been recognized as the most common infectious agent of eosinophilic meningitis or meningoencephalitis (EM) worldwide. Recently, cases of angiostrongyliasis has increased rapidly due to the extension of the natural focus of angiostrongyliasis and the change of human dietary patterns..The parasitologic detection rate of A. cantonensis infection in humans was usually very low. Therefore, serological techniques are considered more sensitive and simple than pathogenic methods for the diagnosis of angiostrongyliasis. So far as we known, the most common immunological methods used in the diagnosis for angiostrongyliasis in this country are IFAT, IEST and ELISA. All antigens used in these methods were prepared from the whole adult worms. Although the sensitivity of these methods is high, the specificity is relative low. Reports indicated that immunological cross-reactions between trichinosis and angiostrongyliasis were observed when the whole worm lysate was used as the antigen. In addition, the whole worm antigens cannot be used to discriminate the acute infection from past infection and to monitor the efficacy of the treatment. In order to develop more sensitive and specific diagnosis systems, we analyze the differences among the antigens of A.cantonensis in different developmental stages and identify dominant diagnostic antigens for Angiostrongyliasis, we also purify the Mr32000 antigen(AC32) from k.cantonensis by electroelution from SDS-polyacrylamide gels and use AC32 to develop the ELISA kit for the diagnosis of Angiostrongyliasis.To analyze the differences among the antigens of Angiostrongylus cantonensis in different developmental stages and identify dominant diagnostic antigens for Angiostrongyliasis .The antigens of A.cantonensis in different developmental stages were analyzed by SDS-PAGE and immunoblot. Results: The protein bands of alldevelopmental stages were similar on SDS-PAGE. The Mr40 000, 50 000, 66 000 and 80 000 antigens reacted not only with the sera of rats infected by A.cantonensis but also with the sera of normal rats. The Mr 104 000 antigen could be discerned by sera of rats infected with A.cantonensis for 2 weeks. The Mr32000 antigen could be recognized by sera of rats 2 weeks after infection, and the reaction became stronger with the infective time lasted. Conclusion: The Mr40 000, 50 000, 66 000 and 80 000 antigens might result in the unspecific reactions in the immunodiagnosis of Angiostrongyliasis using the crude antigen of A.cantonensis. The Mr 104 000 of larva, Mr33 000 of females and Mr32 000 of all worms can be used as candidate antigens in early diagnosis and epidemiological survey of Angiostrongyliasis.To evaluate the specificity and sensitivity of Mr32 000 antigen(AC32) in the immunodiagnosis of Angiostrongyliasis cantonensis. A major antigenic protein of Mr32 000 was purified from A.cantonensis by electroelution from SDS-polyacrylamide gels. AC32-ELISA and adult worm antigen (AWA)-ELISA were used to detect the specific IgG in sera of normal rats and rats with Angiostrongyliasis, sera of healthy individuals and patients with Angiostrongyliasis, schistosomiasis and other parasitosis. Results: The positive rate of sera of rats with Angiostrongyliasis was 100% by AC32-ELISA. There is no psedopositive or cross reaction between the AC32 and all the sera of the normal control and the individuals infected with other parasites but A.cantonensis. Conclusion: The Mr32 000 antigen of A.cantonensis is a valuable candidate in the immunodiagnosis of Angiostrongyliasis.