For ELISA, polystyrene plates were coated overnight with poly- L-lysine (Sigma). For anti-nucleosome ELISA, they were then incubated with ethanol-extracted and S1 nuclease-treated dsDNA from calf thymus (Sigma), followed by an incubation with calf thymus histones type II-AS (Sigma). For anti-RNA ELISA, the plates were incubated with yeast RNA (Sigma). Serial dilutions of serum from 1/200 to 1/5400 were added, after blocking with 1% BSA in PBS. Specific Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology) or IgG2a (Southern Biotechnology). The nucleosome-specific antibody clone PL2–3 and the monoclonal antibody BWR4 were used as standards for the anti-nucleosome and anti-RNA measurements respectively13 (link), 66 . ELISA data were recorded with SpectraMax ELISA Reader and analyzed with DeltaSoft JV (v2.8.11).
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Enzyme-Linked Immunosorbent Assay for Nucleosome and RNA Detection
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Other organizations : University of Pittsburgh, Yale University, The University of Tokyo, Tokyo University of Science
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Variable analysis
independent variables
- Serum dilution (from 1/200 to 1/5400)
- Anti-nucleosome antibody levels
- Anti-RNA antibody levels
- Polystyrene plates coated with poly-L-lysine
- Plates incubated with ethanol-extracted and S1 nuclease-treated dsDNA from calf thymus
- Plates incubated with calf thymus histones type II-AS
- Plates incubated with yeast RNA
- Blocking with 1% BSA in PBS
- Alkaline phosphatase-conjugated goat anti-mouse IgG or IgG2a used for detection
- Nucleosome-specific antibody clone PL2–3 and monoclonal antibody BWR4 used as standards
- Nucleosome-specific antibody clone PL2–3 used as standard for anti-nucleosome ELISA
- Monoclonal antibody BWR4 used as standard for anti-RNA ELISA
- Not explicitly mentioned
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