Research Scientist
Department of Neurology
University of Texas Southwestern Medical Center Dallas
The polyclonal goat-anti-mouse isotype antibodies, IgG1, IgG2a, IgG2b, IgG2c, IgG3, as well as IgA and IgM, come with a number of different labels. For an ELISA assay to determine the isotype in a given mouse plasma or serum sample, three different labels are useful: alkaline phosphatase (AP), horseradish peroxidase (HRP), and biotin (BIOT).
The usual ELISA assay: Antibodies in mouse plasma or serum are measured according to standard procedures. 96-well plates are coated with the respective antigens in coating buffer and incubated overnight at 4°C. Following washes in PBS /0.05% Tween (PBST) buffer, wells are blocked with 1.5% BSA in PBS. Plasma samples are diluted in PBS at concentrations ranging from 1:200 to 1:2000 and incubated again overnight at 4°C. To determine the respective antigen-specific antibody isotype, a second antibody incubation step is now included in the ELISA protocol. The goat-anti-mouse IgG isotype antibodies, IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, and/or IgM are used in dilutions of 1:1000 according to the manufacturer’s instructions and incubated with the samples for 1 hour at room temperature. The detection of these secondary antibodies is performed according to the format used: AP and HRP labeled antibodies are directly used in the substrate reaction (ABTS, TMB) at this step; the biotinylated antibodies are detected after a tertiary incubation step using avidin-conjugated HRP (1 h incubation) which is then developed in the next step via a substrate reaction (ABTS, TMB). Absorbance values from these reactions are read with an ELISA reader.
These antibodies can be also used in B cell ELISPOT assays to determine isotypes and numbers and frequencies of antibody secreting antigen specific B cells. In these assays, higher antibody concentrations are recommended, I use them in a 1:250 dilution with reliable results.