Millex ha 0.45 mm filter

ctDNA was passed through an Millex-HA 0.45 mm filter (Millipore) to eliminate ssDNA. Indicated amounts of ctDNA or supercoiled plasmid DNA were incubated with recombinant human topo I in 40 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.5 mM EDTA, 30 µg/ml BSA for 30 min at 37 °C. Then, 30 µl of sample per well was used for coating a 384-well Nunc MaxiSorp plates overnight at 4 °C. Plates were blocked with PBS containing 1% casein (Pierce) for 1 h at 37 °C, and plates were incubated with monoclonal anti-dsDNA abs at a concentration of 1 µg/ml for 1 h at 37 °C. Subsequently, plates were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech) for 1 h at 37 °C and developed with p-nitrophenol phosphate (Sigma-Aldrich). Optical density (OD) was measured at 405 nm with a reference filter at 490 nm. When used oligonucleotides for these experiments, forward and reverse strands were annealed for 3 min at 93 °C. After incubation with recombinant topo I as described above, modified oligonucleotides were attached to Nunc MaxiSorp plates overnight at 4 °C using DNA Coating Solution from Pierce. Detection of anti-dsDNA binding was performed as described above.

Ninety-six-well Nunc MaxiSorp plates were coated with 5 μg/ml goat anti-mouse IgG (Sigma) or anti-mouse IgM (SouthernBiotech) overnight at 4 °C. Plates were blocked with 1 % bovine serum albumin (BSA) in PBS for 1 h at 37 °C followed by incubation with diluted plasma samples for 1 h at 37 °C. Subsequently, plates were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG or anti-mouse IgM (SouthernBiotech) for 1 h at 37 °C and developed with p-nitrophenyl phosphate (Sigma-Aldrich). Optical density was measured at 405 nm with a reference filter at 490 nm. Sample concentrations were calculated using a standard curve of purified mouse IgG (Sigma) and IgM (SouthernBiotech).
To determine anti-dsDNA antibodies, calf thymus DNA (Invitrogen) was passed through a Millex-HA 0.45-mm filter (Millipore) to remove any ssDNA fragments. Maxisorp plates were half-coated with 100 mg/ml calf thymus DNA in PBS overnight at 4 °C. Plates were blocked with PBS containing 1 % BSA for 1 h at 37 °C. Diluted plasma samples were incubated at 37 °C for 1 h. Bound anti-dsDNA autoantibodies were detected as described above for total IgG or IgM ELISA, respectively.