DEVELOPMENT OF RECOMBINANAT ANTIGEN BASED ELISA FOR SERODIAGNOSIS OF TOXOPLASMOSIS IN HUMANS AND ANIMALS
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Date
2018
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Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu
Abstract
The present study deals with expression of a molecular target of Toxoplasma gondii viz., microneme 10 (MIC10) with assesement of its diagnostic potential in ELISA. The open reading frame (ORF) of MIC10 of RH strain of the Toxoplasma gondii was PCR amplified, ligated with the pET32b expression vector, transformed into DH5 alpha cells for cloning and subsequently expressed using BL21 codon plus competent cells. Recombinant protein was purified to homogeneity using Nickel chelating affinity chromatography and was assessed for its biological activity. Immunoreactivity of the expressed protein was confirmed by westernblot analysis. Checker board titration was used for standardization of ELISA using sera of experimentally infected mice and recombinant MIC10 antigen. The quantitative titre for IgG antibody in ELISA for the known positive and known negative sera was calculated statistically using the cut off value for the optical densities (OD). The mean of OD values of known negative sera plus standard deviation was the cut off value. The sensitivity and specificity of ELISA was generated for mice, goats and humans using the available known positive and negative sera and was 79.2%, 100%, 100% and 87.2%, 87.5%, 100%, respectively. Mature recombinant MIC10 was also used as antigen in ELISA for seroprevalence study on 133 field sera samples from goats and 100 sera samples from humans. The sero-prevalence of 18.79% and 4% in goats and humans, respectively was observed. From the present study, it can be infered that the recombinant MIC10 based ELISA may be used for diagnosis of T. gondii infections in animals and humans.
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No. of references 130