Lse digital microplate shaker

PLGA-DRV formulation (1 mL) was mixed with 4 mL of acetone for at least 2 h at room temperature to extract DRV for measurement of encapsulation efficiency. To determine the drug loading capacity, PLGA-DRV formulation (1 mL) was lyophilized using the Labconco Freeze Dry System overnight (−48 °C, 133 × 10⁻³ mBar; Labconco, Kansas City, MO, USA). Lyophilized PLGA-DRV formulation was reconstituted in 4 mL acetone to extract DRV for the drug loading. To ensure thorough extraction of DRV from the nanoparticles, the acetone solution was placed on a shaker (Corning, LSE Digital Microplate Shaker, Tewksbury, MA, USA) and gently shaken for 24 h at 80 rpm at room temperature to completely extract DRV from the nanoparticles. The supernatant was collected and diluted (1:300) in acetonitrile to measure the concentration in LC-MS/MS.
$$\mathrm{E}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y} (\%)=\frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{i}\mathrm{n} \mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{f}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{s}}\times 100$$
$$\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g} (\%)=\frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{i}\mathrm{n} \mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}\times 100$$

The levels of antibodies against SARS-CoV-2 nucleocapsid and spike proteins were estimated as previously described [38 (link)]. Beads coupled to SARS-COV-2 nucleocapsid and spike, following a protocol(hal-01299922 [39 ] (courtesy of National Research Institute for Sustainable Development, France, https://en.ird.fr/), were used in this study. To eliminate cross-reactive responses, nucleocapsid and spike antigen from SARS-CoV-1 and MERS-CoV were also included. The coupled beads (25 μl of each bead) were distributed into a 96-well plate. The supernatant was removed while beads were mobilized using a magnetic holder and blood plasma (1:100) was added to the plate. The plate was incubated for 2 h at 25°C on a plate shaker (400 rpm), then washed. An anti-human lgG secondary antibody (4μg/ml) was added to the plate and incubated for 30 min in a Corning® LSE™ digital microplate shaker (400 rpm). This was followed by 3 washes, addition of streptavidin-phycoerythrin (1μg/ml) (Invitrogen) and incubation for 10 min in a plate shaker (400 rpm). Finally, the plates were read using a Luminex MAGPIX system (Luminex Corporation, Austin, TX, USA) using xPONENT™ software (V.4.3.229).

The levels of antibodies against SARS-CoV-2 nucleocapsid proteins were quantified as previously described [20 (link), 21 (link)]. SARS-COV-2 nucleocapsid protein was coupled to Luminex beads, following an established protocol (hal-01299922 [22 ] courtesy of National Research Institute for Sustainable Development, France, https://en.ird.fr/). A 25 µL solution containing coupled beads was aliquoted into each well of a 96-well plate. The supernatant was removed while beads were immobilized using a magnetic holder and 1/100 diluted blood plasma was then added to each well. The plate was incubated for 2 h at 25 °C on a plate shaker at 400 rpm, then washed three times with wash buffer. Anti-human lgG secondary antibody (4 µg/mL) was added to the plate and incubated for 30 min on a Corning^® LSE™ digital microplate shaker at 400 rpm. This was followed by three washes, the addition of 1 µg/mL streptavidin–phycoerythrin (Invitrogen) and incubation for 10 min on a plate shaker at 400 rpm. Finally, the plates were read on a Luminex MAGPIX system (Luminex Corporation, Austin, TX, USA) using xPONENT™ software (V.4.3.229).