Before performing the complement activation assay, the levels of endotoxin contamination in the selected liposomes were quantified as presented in our recent work.19 (link) Briefly, the endotoxin levels were determined using the Limulus amoebocyte lysate (LAL) chromogenic methods by kinetic assay. The control standard curve was prepared with the standard endotoxin from the E. coli O111:B4 strain (cat. EC010, 10 ng/vial; Associates of Cape Code, Inc.). The sample readouts were carried out at 405 nm, according to manufacturer’s instruction. Results for each sample were taken into consideration only if the correlation coefficient of the calibration curve was ≥0.98.
A previously published protocol for the quantitative determination of complement activation in human plasma was further optimised as described inFigure 2 .20
The liposome’s stock solutions were stored at 4 °C. Working solutions of 450 μg/mL and 150 μg/mL were prepared by diluting the liposomes in sterile PBS buffer (pH 7.4). The liposome solutions for in vitro testing were prepared at concentrations of 50 and 150 μg/mL by mixing the liposomes working solutions with PBS and human serum. In brief, the enzyme-linked immunosorbent assay (ELISA) was performed according to the MicroVue iC3b EIA Quidel manufacturer’s instructions. Serum samples were diluted in a U-shaped 96-well plate by 1:100 in the specimen diluent. Then the standards, the controls and the diluted serum samples were transferred to the ELISA plate and incubated at room temperature for 30 min. The ELISA plate was thoroughly washed with wash solution provided by the kit. The iC3b conjugate was added to the plate for 30 min and after washing steps, substrate solution was added to the plate for 30 min. Finally, the addition of stop solution blocked the enzymatic reaction and the iC3b levels were determined by absorbance reading at 405 nm with an EnSpire® Multimode plate reader (Perkin Elmer).
A previously published protocol for the quantitative determination of complement activation in human plasma was further optimised as described in
The liposome’s stock solutions were stored at 4 °C. Working solutions of 450 μg/mL and 150 μg/mL were prepared by diluting the liposomes in sterile PBS buffer (pH 7.4). The liposome solutions for in vitro testing were prepared at concentrations of 50 and 150 μg/mL by mixing the liposomes working solutions with PBS and human serum. In brief, the enzyme-linked immunosorbent assay (ELISA) was performed according to the MicroVue iC3b EIA Quidel manufacturer’s instructions. Serum samples were diluted in a U-shaped 96-well plate by 1:100 in the specimen diluent. Then the standards, the controls and the diluted serum samples were transferred to the ELISA plate and incubated at room temperature for 30 min. The ELISA plate was thoroughly washed with wash solution provided by the kit. The iC3b conjugate was added to the plate for 30 min and after washing steps, substrate solution was added to the plate for 30 min. Finally, the addition of stop solution blocked the enzymatic reaction and the iC3b levels were determined by absorbance reading at 405 nm with an EnSpire® Multimode plate reader (Perkin Elmer).
