Quantiscan 3

Assays to detect anti-leishmanial antibodies were carried out at room temperature, by applying 70 μl of diluted serum to the sample well. For the analysis, samples were thawed at room temperature, carefully mixed and diluted by 1:20 using the running buffer (phosphate buffer 20 mM, pH 7.4, 50mM NaCl, 1% BSA, 0.5% PVA, 0.1% Triton X-100).
Qualitative results were judged by the naked eye after 15 minutes (Fig. 2). Samples were analysed in duplicate and results were observed by three operators. Images of LFIA devices were also acquired by a portable scanner (OpticSlim 550 scanner, Plustek Technology GmbH, Norderstedt, Germany) and the area of the coloured lines was quantified by means of the QuantiScan 3.0 software (Biosoft, Cambridge, UK).

The fluorescent ICST in the dipstick format was carried out at room temperature. 95 µl of sample extracts (or FMB1 standards prepared as described above) were transferred into wells of a 96-well black microtiter plate (VWR International, Milan, Italy) and we added 5 µl of QD-Ab conjugate. To start the capillary migration process, the strip was dipped in the well after homogenization of the solution by gently pipetting (Figure 1). After 10 min, the strip was removed and dried for 5 min at 37°C before observing the fluorescence signal. The test results were both qualitatively estimated by the naked-eye under a UV light at 365 nm (Camag, Berlin, Germany) in a dark room, and quantitatively evaluated using a super chargecoupled device camera (Fujifilm, Tokyo, Japan) and the digital processing of images with QuantiScan 3.0 software (Biosoft, Cambridge, UK). Before data processing, we applied a red filter on the images, in order to subtract the signal on the lines due to the fluorescence of proteins. The quantitative information was obtained processing the negative of the Tagged Image File Format. To evaluate assay results, the signals from T-line and control line (C-line) for each test were processed.
Standard calibration curves were obtained by plotting the T-line/C-line ratio versus FMB1 standard concentrations and fitting data with a four-parameter logistic equation.