GMC20 project protocol

Circulating immunoglobulins, rheumatoid factor, and anti-DNA antibodies in 8 inbred founder strains of the Collaborative Cross   (2020)

German Mouse Clinic and Department of Infection Genetics, HZI
With: Hrabě de Angelis M, Fuchs H, Gailus-Durner V, Adler T, Treise I, Busch D, Aguilar-Pimentel A, Ollert M, Lengger C, Kollmus H, Schughart K




German Mouse Clinic: Phenotyping Pipeline

Immunoglobulin and autoantibody assessment: 16-17 and 20-21 wks of age

 

  • Project protocol - Contents

    Workflow and sampling

    Step
    Procedure
    		 Equipment
    Data collected
    1 Body weight Balance Body weight
    2 Mice anesthetized - -
    3 Immunoglobulin and autoantibody assessment ELISA reader Plasma immunoglobulin levels, anti-DNA autoantibody level, rheumatoid factor level

    Equipment and supplies

    • ELISA reader: Biorad (Bio-Plex System), USA

    Reagents and solutions

    • Isoflurane
    • 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

      1. Mice are anesthetized by isoflurane and blood collected by puncturing the retro-orbital plexus.
      2. 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.
      3. 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.
      4. 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.

    Data collected by investigator

    • Plasma IgM
    • Plasma IgA
    • Plasma IgG1
    • Plasma IgG2a
    • Plasma IgG2b
    • Plasma IgG3
    • Plasma IgE
    • Plasma anti-DNA autoantibodies
    • Plasma rheumatoid factor
    		•