ELISAs were performed using 96 well Nunc-Immuno MaxiSorp plates (Thermo Fisher) coated with dsDNA (Sigma-Aldrich), phosphorylcholine (PC)-10 (Sigma-Aldrich), Sm/RNP (Arotec Diagnostic), or Qβ-VLP (1μg/ml in PBS) (23 (link)). Plates were blocked with 1% BSA in PBS prior to incubation with diluted serum or supernatant. Specific antibodies were detected using goat anti-mouse IgM-, IgG-, or IgG2c -HRP (SouthernBiotech) and peroxidase reactions were developed using OptEIA TMB substrate (BD Biosciences) and stopped with 2N H2SO4. Absorbance at 450nm was read using a SpectraMax 190 microplate reader (Molecular Devices) and data analyzed using GraphPad Prism (GraphPad Software, Inc.). Autoantigen microarrays were performed at the UT Southwestern Medical Center Microarry Core Facility, Dallas, TX (24 (link)).
Protocol detail
Autoantigen ELISA and Microarray Protocols
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Anti igm
Antibodies
Autoantigen
Devices
Diagnostic
Dsdna
Elisas
Goat
Microarrays
Mouse
Peroxidase
Phosphorylcholine
Prism
Serum
Corresponding Organization : University of Washington
Other organizations : King Abdulaziz City for Science and Technology, The University of Texas Southwestern Medical Center, Institute of Biophysics, Chinese Academy of Sciences
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Variable analysis
independent variables
- DsDNA (Sigma-Aldrich)
- Phosphorylcholine (PC)-10 (Sigma-Aldrich)
- Sm/RNP (Arotec Diagnostic)
- Qβ-VLP (1μg/ml in PBS)
- Specific antibodies detected using goat anti-mouse IgM-, IgG-, or IgG2c -HRP (SouthernBiotech)
- Absorbance at 450nm
- 96 well Nunc-Immuno MaxiSorp plates (Thermo Fisher)
- 1% BSA in PBS
- OptEIA TMB substrate (BD Biosciences)
- 2N H2SO4
- Not explicitly mentioned
- Not explicitly mentioned
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