Biotin rat anti mouse igm r6 60

Quantification of immunoglobulin and isotyping were done by ELISA; coating 96-well microtiter plates (Costar) with 3 µg/mL of F(ab’)₂ Fragment Goat Anti-Mouse IgM, µ chain specific (Jackson ImmunoResearch) or Goat anti-mouse IgG (Sigma) polyclonal antibodies. Hybridoma supernatants were diluted 1/2 for the determination of antibody production; and 1/20 for IgG subclass determination. Production and isotype determination were tested with Biotin Rat anti-mouse IgM (R6-60.2, BD Biosciences) and Horseradish Peroxidase (HRP)-conjugated Goat anti-mouse IgG (Sigma). IgG subclasses determination was done using Biotin anti-mouse IgG1, IgG2b, IgG2c and IgG3 polyclonal antibodies (Jackson ImmunoResearch). HRP-conjugated streptavidin (Roche) was used for biotinylated antibodies detection. Finally, microplates were developed with TMB (BD Bioscience) and reaction was stopped with 2M H₂SO₄. Microplates were read with an EPOCH Microplate Spectrophotometer (BioTek) at 450-570 nm.

Reactivity determination was done using high binding plates (Costar) coated with the following antigens at 3 μg/mL: calf thymus double-stranded DNA (Sigma); Histone (H1; Sigma); Ro52 (SSA-TRIM21; Sigma); LPS from Escherichia coli O55:B5 (Sigma); human Insulin (Lilly) and Ovalbumin (InvivoGen). Supernatants of highly confluent hybridoma cultures were diluted 1/2; HRP-conjugated goat anti-mouse IgG (Sigma) polyclonal antibody and Biotin rat anti-mouse IgM (R6-60.2, BD Biosciences) were used as detection antibodies and HRP-conjugated streptavidin (Roche) for the biotinylated antibody detection. Finally, microplates were developed with TMB (BD Bioscience) and reaction was stopped with 2M H₂SO₄. Microplates were read with an EPOCH Microplate Spectrophotometer (BioTek) at 450-570 nm. Antibodies which were positive for three or more antigens of our panel were considered polyreactive.