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A dot enzyme-linked immunosorbent assay was standardized using excretory-secretory antigens of Toxocara …


Biology Articles » Biochemistry » Enzymology » Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis » Discussion

Discussion
- Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

The development of specific, sensitive, and reliable techniques to demonstrate the presence of antibody in toxocariasis is an important step towards improving diagnosis. In this study, we evaluated a simple and sensitive dot-ELISA test for the immunodiagnosis of human toxocariasis. Conventional ELISA procedures require larger volumes of soluble antigen (De Savigny et al. 1979, Espinoza et al. 2003), whereas the dot-ELISA uses only minute amounts of TES antigen per test (0.2 ng total protein per dot in contrast to 1.25 µg per microtitration well). We found that the best result to fix TES antigen to NC sheets was obtained with sodium bicarbonate buffer with 2% OVA which could be responsible for the need for lesser antigen concentration than previously reported (Camargo et al. 1992). Simplification of the procedure can be achieved by the use of large NC strips (Boctor et al. 1987), mass production of NC strips (Janitschke et al. 1987), and the use of the commercially available Bio-dot apparatus (Chan & Ko 1988). Another advantage of this method is that it is simple, rapid (about 2 h), does not need expensive equipment, results can be read visually, a large number of samples can be assayed, and the test is performed at room temperature.

The sensitivity of dot-ELISA and standard ELISA was similar (100%) as previously reported (Camargo et al. 1992), whereas specificity of the dot-ELISA was higher than standard ELISA. The healthy control sera were negative by both tests and other parasites-positive group, 3 out of 20 sera gave a positive reaction in the dot-ELISA and 6 out of the same subjects were positive for ELISA. The positive reactions observed specially with ELISA was undetermined but may include the presence of common antigens between Toxocara antigens and several other infectious agents commonly occurring in underdeveloped countries and previous unapparent past infections with Toxocara species. Previous absorption of sera with A. suum antigen extract, does not eliminate all cross-reactions, as reported (Camargo et al. 1992).

ELISA is the most commonly utilized diagnostic serologic test for VLM, OLM, and covert toxocariasis, the most frequent clinical syndromes associated with Toxocara infections. However, the serum ELISA for Toxocara-specific IgG is less sensitive for the diagnosis of OLM than for other forms of the disease (Magnaval et al. 2001, Despommier 2003). Therefore, confirmation of ocular toxocariasis can be obtained by testing aqueous or vitreous fluid. Dot-ELISA here presented has not yet been performed with sera or intraocular fluids of patients with OLM but we think this test will not only improve the diagnosis of VLM because its specificity is higher than the indirect ELISA but also the diagnosis of ocular toxo-cariasis.

The dot-ELISA for toxocariasis, as reported here, is a specific means of establishing serological diagnosis of the disease. The dot-ELISA can be used as a qualitative test to screen large number of samples and can be further developed as a field detection method.

ACKNOWLEDGEMENTS

To Dr Hilda Solís, Instituto de Medicina Tropical " Daniel A. Carrión ", for kindly supplying the nitrocellulose sheets.

 


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