#Primary antibody cross reactivity series

If a goat is immunized with the mouse IgG, it will produce goat anti-mouse IgG antibodies, which will bind to all fragments and classes of IgG. Here’s an example for better understanding. Thus, it’s essential to understand which antibody will be suitable for your experiment.įigure: An illustrative diagram of the production of secondary antibodies in different hosts. Secondary antibodies are raised in different animal species and conjugated with different enzymes and fluorophores.

#Primary antibody cross reactivity series

A series of subclasses of these antibodies are available to carry out specific lab procedures. The five classes of secondary antibodies include IgG, IgM, IgD, IgE, and IgA antibodies. Thus, if you choose a mouse IgG1 monoclonal primary antibody for your workflow, an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG will be required for the assay. Similarly, monoclonal antibodies are raised in animal species like rabbits, mice, and rats. Thus, an anti-IgG H&L (Heavy & Light chains) antibody will be the preferable antibody class for those workflows. The polyclonal primary antibodies are generally IgG isotype and raised in host animals, such as goats, sheep, rabbits, and donkeys.

They are specifically designed to suit a variety of workflows. Classes of Secondary Antibodies and Choosing the Right OneĬommercially, more than 90 different types of secondary antibodies are available for R&D purposes. The secondary antibody used in the workflow must have specificity for the antibody host species and the isotype of the primary antibody.īecause of the involvement of both primary and secondary antibodies and the fact that multiple secondary antibodies bind to a single primary antibody, the indirect method helps increase the signal amplification and sensitivity of an assay.įigure: An illustration of the difference between direct and indirect immunoassays. Then, secondary antibodies bind to the primary antibodies. Here, the membrane/surface is first incubated with the primary antibody that binds to the target protein. Indirect detection involves two incubation steps. However, this method is less sensitive, and has poor reproducibility and increased background. It reduces the cross-reactivity between different species (other than the target). Here, the labeled primary antibody is added to the membrane, and the protein is detected only after a single incubation step.

Direct Detectionĭirect detection only requires one labeling step. The fluorophore attached to secondary antibodies helps in studying the localization and distribution of the protein of interest or target antigen using the fluorescent microscopy technique.Īnd, based on the attachment of a label to the primary or secondary antibody, the detection method is of two types: direct and indirect detection technique. See Equipment List Request Estimate How Do Secondary Antibodies Work?