German Mouse Clinic: Phenotyping Pipeline
Immunoglobulin and autoantibody assessment: 16-17 and 20-21 wks of age
||Immunoglobulin and autoantibody assessment
||Plasma immunoglobulin levels, anti-DNA autoantibody level, rheumatoid factor level
- ELISA reader: Biorad (Bio-Plex System), USA
- Monoclonal antibodies to all immunoglobulins except IgE: Biorad, USA
- Anti-mouse-IgE rat monoclonal IgG (clone-PC284, The Binding Site)
- Rabbit IgG: Sigma-Aldrich, Steinheim GERMANY
- Calf thymus DNA: Sigma-Aldrich, Steinheim GERMANY
- AP-conjugated goat anti-mouse secondary antibody: Sigma-Aldrich, Steinheim GERMANY
- Positive control in autoantibody assays: MRL/MpJ-Fas<lpr>/J mice (The Jackson Laboratory, Bar Harbor ME)
Procedure: Immunoglobulin and autoantibody assessment
- Mice are anesthetized by isoflurane and blood collected by puncturing the retro-orbital plexus.
- Plasma levels of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA are determined simultaneously in the same sample using a bead-based assay with monoclonal anti-mouse antibodies and a Bioplex reader.
- The presence of rheumatoid factor and anti-DNA antibodies are evaluated by indirect ELISA with rabbit IgG and calf thymus DNA, respectively, as antigens and AP-conjugated goat-anti-mouse secondary antibody (serum samples from MRL/MpJ-Fas<lpr>/J are used as positive controls). Note that both parameters are measured semi-quantitatively (values are optical densities, e.g., readouts from the ELISA) therefore, the values are not compared to a standard curve and do not represent actual concentrations; they are unitless.
- Plasma is analyzed for total IgE using a classical immunoassay isotype-specific sandwich ELISA where 96-well plates are coated with 10 µg/mL anti-mouse-IgE rat monoclonal IgG to detect total IgE. Plates are analyzed at 450 nm.
- Plasma IgM
- Plasma IgA
- Plasma IgG1
- Plasma IgG2a
- Plasma IgG2b
- Plasma IgG3
- Plasma IgE
- Plasma anti-DNA autoantibodies
- Plasma rheumatoid factor