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Importance of isotype controls

The specificity of staining by monoclonal antibodies to target antigens should be verified by establishing the amount of non-specific antibody binding. Especially at higher concentration (more than 10 µg/ml) the antibody staining usually has consignable background. To this end a non-reactive immunoglobulin of the same isotype is included as a negative control for each specific monoclonal antibody used in a particular immunoassay. Such an isotype control antibody is usually generated to an irrelevant antigen, and does not cross-react with species of interest, hence all the background that could be observed when working with this antibody would be a result of general nonspecific interactions between an immunoglobulin molecule and the respective sample under the particular conditions. This shall help the customer to set up the experimental conditions so that the nonspecific binding of any antibody is abolished, or at least to take in account the level of background signal.

Currently available isotype controls in our portfolio:

Fig.1: Flow cytometry surface nonspecific staining pattern of human peripheral whole blood stained using mouse IgG2a Isotype control Alexa Fluor® 488 antibody (A4-724-C100, concentration in sample 9 μg/ml).

Fig.2: Flow cytometry surface nonspecific staining pattern of human peripheral whole blood stained using rat IgG2b Isotype control FITC antibody (1F-169-C100, concentration in sample 9 μg/ml).