Abstract
BackgroundThe inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated.MethodsAn indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.ResultsThe diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.ConclusionsThis finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.
Highlights
The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents
The immunoreactivity of USM.TOXO1 antigen against anti-T.gondii IgM antibodies was confirmed by western blot and Dot enzyme immunoassay analysis
USM.TOXO1 recombinant proteins by indirect enzyme-linked immunosorbent assay (ELISA) To evaluate the potential of USM.TOXO1 antigen for the detection of anti T. gondii IgG antibodies in human sera, an in-house ELISA was developed using USM.TOXO1 fusion proteins as capture antigen
Summary
Serum samples Hospital Universiti Sains Malaysia (HUSM) of Kelantan is situated at the north east of peninsular Malaysia. The optimal concentration of the coating antigen and the serum, conjugate dilution were determined by checkerboard titration assay using known positive and negative human sera. The following day the wells were washed (3X) with PBS-T for 5 min each time and blocked with 200 μl of blocking buffer for 1 h at 37 °C After another rounds of washing, 100 μl of human sera diluted at 1: 400 was added to the wells and incubated at 37 °C for 1 h. At the end of the incubation time the wells were washed again and 100 μl of HRP conjugated anti-human IgG antibody (diluted 1:4000) was added for 1 h at 37 °C, followed by final 3X wash. The immunoreactivity of USM.TOXO1 antigen against anti-T.gondii IgM antibodies was confirmed by western blot and Dot enzyme immunoassay (dot-EIA) analysis. Statistics The sensitivity, specificity, negative, and positive predicted values were calculated by MedCalc online Software, using the following formulas: sensitivity: TP/TP + FN × 100%; specificity: TN/TN + FP × 100%; positive predicted value: TP/TP + FP × 100%; negative predicted value: TN/TN + FN × 100, where TP is the number of true positive; FP, the number of false-positive; FN, the number of false negative; and TN, the number of true negative
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