Autoreactivity against nuclear and cytoplasmic self-antigens was determined with QUANTA Lite ANA ELISA (Inova Diagnostics) as described (52 (link)). Polyreactivity against ssDNA, dsDNA, Keyhole limpet hemocyanin (KLH), human insulin and lipopolysaccharide (LPS) was determined as described (22 (link)). To measure NP binding, high-binding 96-well plates (Corning) were coated overnight with 50 μl of PBS containing 10 μg/ml of NP4-BSA or NP25-BSA (Biosearch Technologies). After washing with PBS containing 0.05% of Tween 20 (Sigma), wells were blocked with PBS containing 1% of BSA for 2 h at room temperature. Monoclonal antibodies were incubated at 4 μg/ml or 7 consecutive 1:4 dilutions in PBS for 2 h at room temperature. After washing, HRP-conjugated goat α-human IgG (Jackson ImmunoResearch) was added at 0.16 μg/ml for 1 h at room temperature. After additional washing, HRP was revealed with 1-Step ABTS Substrate Solution (ThermoFisher Scientific). Absorbance was measured at 405 nm after incubation for 20 min at room temperature.
Protocol detail
Measurement of Antibody Binding to NP-BSA Conjugates
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QUANTA Lite ANA ELISA
high-binding 96-well plates
NP4-BSA
NP25-BSA
Tween 20
1-Step ABTS Substrate Solution
Abts
Cytoplasmic
Diagnostics
Dilutions
Dsdna
Elisa
Goat
Human
Insulin
Keyhole limpet hemocyanin
Lipopolysaccharide
Monoclonal antibodies
Self antigens
Ssdna
Tween 20
Corresponding Organization :
Other organizations : Rockefeller University, University of Amsterdam, Academic Medical Center, Cornell University, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Concentration of monoclonal antibodies (4 μg/ml or 7 consecutive 1:4 dilutions)
- Absorbance at 405 nm (after incubation for 20 min at room temperature)
- Coating of high-binding 96-well plates with 50 μl of PBS containing 10 μg/ml of NP4-BSA or NP25-BSA
- Washing of wells with PBS containing 0.05% of Tween 20
- Blocking of wells with PBS containing 1% of BSA for 2 h at room temperature
- Incubation time of monoclonal antibodies (2 h at room temperature)
- Concentration of HRP-conjugated goat α-human IgG (0.16 μg/ml)
- Incubation time of HRP-conjugated goat α-human IgG (1 h at room temperature)
- Use of 1-Step ABTS Substrate Solution (ThermoFisher Scientific) to reveal HRP
- Positive control: Not specified
- Negative control: Not specified
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