Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes

摘要

Objective: To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex-(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum. Methods: The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. Results: The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m2 were put in contact with undiluted sera in buffer borate pH 8-20 mm containing glycine (0.1 m) and polyethyleneglycol 8000 (3% w/v). Finally, the latex-protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. Conclusion: The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI.