Screening Diagnostic Antigen Of Toxoplasmosis Based On Proteomics Technology

ObjectiveToxoplasma gondii (T. gondii) is an obligatory intracellular parasite that infects almost one third of people worldwide. Since the vagaries associated with lack of specificity, sensitivity, and not reliability of many commercial serologic test kits on the market, there is no reliable evidence in clinical diagnosis and treatment. In an attempt to discover useful candidates for the diagnosis of toxoplasmosis, proteomics in combination with western blotting were employed in this study. Furthermore, assess its role in the diagnosis of toxoplasmosis.MethodsFollowing two dimensional electrophoresis (2-DE), the tachyzoite lysates of T. gondii RH strain were transfered onto NC membranes for Western-blot analysis. The developed films were aligned and then matched with the homologous Coomassie brilliant blue (CBB)-stained gels by PDQuest software and artificial recognition. The matched spots were excised, digested by trypsin and further analyzed by LC/MS-MS. The potential biomarkers of the identified protein was cloned and expressed from the transformed E. coli. The recombinant was purified, coated in micro-well plates and evaluated their performance in diagnosis of toxoplasmosis using enzyme linked immunosorbent assay (ELISA). Besides, the BALB/c mice were orally infected with cysts of PRU strain of T. gondii and bled to obtain sera at different time points for specific IgG and IgM detection.ResultsThis serological proteome assay yielded more than 50 immunodominant spots. 21 of these spots in Toxo-IgM^–-IgG⁺ group were precisely matched with a homologous two-dimensional electrophoresis (2-DE) gel and successfully identified by LC/MS-MS as corresponding to 13 different proteins. However, pooled sera with Toxo-IgM⁺-IgG^– showed just 3 spots could be matched to 2-DE gel perfectly, and only one of them could be identified and characterized by LC/MS-MS: the rhpotry protein 2 (ROP2). Of these proteins, rROP2186–533 was successfully expressed and further applied in the diagnosis of human toxoplasmosis using ELISA. The ELISA results revealed sensitivities of 100% and 0% for anti-Toxo IgM and IgG antibodies, respectively. Furthermore, the assays showed a specificity of 100% for IgM antibody test. There was a significant difference of OD values of Toxo-IgM between the sera of Toxo-IgM⁺-IgG^– and control (P﹤0.0001).Blood samples from the mice infected with the PRU strain were detected by rROP2186–533, data showed that The Toxo-IgM antibodies appeared on day 7 post-infection (PI), peaked on 14 PI and rapidly decreased on 28 PI when tested with rROP2_{186–533} antigen. The data showed that the kinetics of Toxo-IgM antibodies response was consistent with that generally accepted. But, the titer of specific IgG antibodies had no significant change before and after infection.ConclusionProtein ROP2 was successfully screened and recombined from the tachyzoite lysates of T. gondii RH strain. Our present study confirmed that rROP2_{186–533} was an useful diagnostic antigen for detecting Toxo-IgM antibody and could clearly distinguish acute and chronic infection. And the immunoproteomic approach has promising potential and offers a powerful tool in the screening of candidates for the diagnosis of toxoplasmosis. Our primary investigation laid the foundation for the development of laboratory reagent with high specificity and sensitivity for the diagnosis of toxoplasmosis.