Spectramax plus 384 system

The fluorescence of the E. coli strains was measured with a Fluoroskan Ascent (Thermo-Labsystems, Helsinki, Finland). The following excitation/emission pairs (in nm) were used: GFP^mut3.1, 485/527, mRFP, 584/620. To correct for variations in cell number, the fluorescence intensities were normalized by the cell density (OD₆₀₀) of the culture measured in transparent 96-well plates with a SpectraMax Plus 384 system (Molecular Devices, Sunnyvale, CA).
Flow cytometric analyses of the cell mixtures, were conducted as follows: immediately after each round of recombineering (or after recovery from the glycerol stock), the cell mixture was grown in LB medium in test tubes (at 37°C at 200 rpm). Then, 20 μL of the overnight cultures was added to 2 mL of fresh LB medium containing 10 mM arabinose and shaken in a test tube at 37°C for 3 h. Approximately 10,000 cells were applied to a MACSQuant VYB flow cytometer (Miltenyi Biotech, Bergisch-Gladbach, Germany) equipped with a 561 nm laser and appropriate filter sets for mRFP (586/15). The data were analyzed by using the MACSQuant Analyzer (Miltenyi Biotech, Bergisch-Gladbach, Germany).

The samples were tested for the presence of Abs against HERV-W_(248–262), IFN-α, and IFN-ω antigens using an indirect enzyme-linked immunosorbent assay (ELISA). Ninety-six-well Nunc immunoplates were incubated overnight at 4°C with 10 μg/mL of the respective peptides in 0.05 M carbonate-bicarbonate (pH 9.5) (Sigma-Aldrich, St. Louis, MO, USA).
Plates were washed with 0.1% Tween 20 in phosphate-buffered saline (PBS-T) and blocked with a solution of 5% skimmed milk for 1 h at room temperature. Plasma samples, diluted 1:100, were added, and the plates were incubated for 2 h. After another washing step, the plates were incubated for 1 h with 100 μL of PBS and an alkaline phosphatase-conjugated goat anti-human IgG polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). Alkaline phosphatase was detected after 8 min of incubation in a solution containing MilliQ water and para-nitrophenyl phosphate (Sigma). The optical density was read at 405 nm using the SpectraMax Plus 384 system (Molecular Devices, Sunnyvale, CA, USA).
Each sample was run in duplicate. Positive- and negative-control plasma samples were also included in all experiments.
A competitive ELISA was performed by using IFN-α, IFN-ω, and a different peptide (annexin A2 positions 13 to 37, LEGDHSTPPSAYGSVKAYTNFDAER, to which the patient was positive) in order to assess the specificity of binding for each peptide.

The samples were tested for the presence of Abs against HERV-W_(248–262), IFN-α, and IFN-ω antigens using an indirect enzyme-linked immunosorbent assay (ELISA). Ninety-six-well Nunc immunoplates were incubated overnight at 4°C with 10 μg/mL of the respective peptides in 0.05 M carbonate-bicarbonate (pH 9.5) (Sigma-Aldrich, St. Louis, MO, USA).
Plates were washed with 0.1% Tween 20 in phosphate-buffered saline (PBS-T) and blocked with a solution of 5% skimmed milk for 1 h at room temperature. Plasma samples, diluted 1:100, were added, and the plates were incubated for 2 h. After another washing step, the plates were incubated for 1 h with 100 μL of PBS and an alkaline phosphatase-conjugated goat anti-human IgG polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). Alkaline phosphatase was detected after 8 min of incubation in a solution containing MilliQ water and para-nitrophenyl phosphate (Sigma). The optical density was read at 405 nm using the SpectraMax Plus 384 system (Molecular Devices, Sunnyvale, CA, USA).
Each sample was run in duplicate. Positive- and negative-control plasma samples were also included in all experiments.
A competitive ELISA was performed by using IFN-α, IFN-ω, and a different peptide (annexin A2 positions 13 to 37, LEGDHSTPPSAYGSVKAYTNFDAER, to which the patient was positive) in order to assess the specificity of binding for each peptide.