After the gel had solidified, 10 μl of each PCR amplification product was mixed with a 2 µl of 6× loading buffer in the well created on agarose gel.
Next , 25 μL of plasma was combined with assay buffer in the well of a 96-well plate.
For all experiments, the final concentration of the protein in the well was ∼15 µg/ml, and the buffer in the well consisted of buffer A (10 mM sodium phosphate, pH 7).
Subsequently 0.5 µg of biotin labeled DBS diluted in 50 µl 1X GMS buffer was added in the well and incubated at 4°C for 1 hour.
Specifically, the MSIA-Tips were mounted onto the head of the Versette and initially rinsed with assay buffer (10 mM PBS w/0.1%TWEEN 20), with 20 cycles (1 cycle consisting of a single aspiration and dispense of a 100 uL volume), from a single 200 uL buffer aliquot placed in the well of a microplate.
Then, the BSA buffer in each well was discarded and the plate was washed three times with PBST buffer (0.05% Tween-20 in PBS buffer).
A biotinylated polyclonal anti-C3 antibody (CC7761, Sigma, St . Louis MO, USA) was diluted in blocking buffer and incubated in the wells for 1 h at RT followed by a streptavidin-HRP conjugate (21130, Pierce, Thermo Scientific, Rockford, IL, USA).
When IFN-α was used as the antigen, 100 μL of a solution of 10 μg/mL of IFN-α in sodium carbonate buffer was placed in the wells, corresponding to 1 μg/well.
Strains were present in duplicate on each plate, with a buffer of WT in the wells around the edge of the plate, so edge effects would not impact test colony measurements.
Then, 100 μl binding buffer containing 10 kBq of In-DOTA-E-[c RGDfK ]2 and approprIn-DOTA-E-[c RGDfK ]20−6–8 × 10−11 M) of Ga(In-DOTA-E-[c RGDfK ]2lled In-DOTA-E-[c RGDfK ]2-E-[c(RGDfK)]2 and DOTappropriatefK)]2}2 in bindilutionser were incubated in the wells at 37°C for 1 h.
A 3-mm slice of each gel plug was incubated overnight at 25°C with SmaI (Life Technology, Invitrogen, Carlsbad, CA, USA) and buffer, then placed in the wells of a 1.0% agarose gel (Ultra pure agarose, Life Technology, Invitrogen), sealed with 1.0 % agarose, and put in 0.5× Tris-borate-EDTA buffer.
