Establishment Of A Sandwich ELISA To Toxoplasma Gondii Circulating Antigen

Toxoplasma gondii, an obligate intracellular protozoan parasite,is capable of infecting a variety of mammals. Humans can be infected by consuming undercooked or raw meat (e.g. pork, lamb) containing tachyzoites or bradyzoites,or through food, water or unwashed vegetables contaminated with oocysts excreted by cats.Women infected with T. gondii before pregnancy usually do not transmit it to the fetus; however, if infection occurs during pregnancy, the pathogen will be transmitted transplacentally to the fetus. Toxoplasmosis in fetuses and neonates may present in various ways, from hydrocephalus, chorioretinitis and intracranial calcifications to hepatosplenomegaly, thrombocytopenia, microcephaly, convulsions, fever and small-for-gestational-age size at birth. Nevertheless, most infected neonates are asymptomatic at birth. Risk of congenital toxoplasmosis is somewhat lower if infection occurs during the first trimester of pregnancy than in the third, but the severity of congenital infection is substantially higher, and primary infection acquired during pregnancy may cause severe damage to the fetus.Therefore, toxoplasmosis is a major public health problem.MIC10 belongs to the microneme protein family and is secreted from micro-organelles, the micronemes, of T. gondii tachyzoites. Mature MIC10, whose function is unknown, is an 18 kDa protein shown to be expressed preferentially in tachyzoites rather than bradyzoites and was previously shown to be released from parasites . Unlike other microneme proteins, MIC10 lacks the transmembrane domain that is capable of binding to host cell receptor(s) and hence this raises the possibilitythat MIC10 could diffuse from the infection site in the tissue and become accessible as a circulating antigen .In this study, obtained through in vitro culture of Toxoplasma gondii parasite , the extraction of T. gondii DNA. Toxoplasma gondii DNA as the template for PCR amplification, sequence of three sections are MIC10A, MIC10B and MIC10C.After cloning, expression and purification, we can get recombinant protein MIC10A-His, MIC10B-His and MIC10C-His. Recombinant protein MIC10A-His and MIC10B-His immunize New Zealand white rabbits, polyclonal antibodies. Purified by conventional methods, namely, affinity chromatography purification of anti-serum, obtained polyclonal antibodies. The rabbit anti MIC10A as coating antibody, biotinylated rabbit anti MIC10B as the detection antibody, the establishment of double antibody sandwich ELISA detection of Toxoplasma gondii circulating antigen MIC10C, established with specificity and sensitivity of experimental methods.