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The secondary antibody is directed against the species of the primary antibody. If you use a primary antibody raised in rabbit, you will need an anti-rabbit secondary antibody raised in a species other than rabbit.
For example, goat polyclonal to rabbit IgG - H&L (HRP) (ab6721) can be used to detect rabbit polyclonal to Ki67 (ab15580).
The secondary antibody has to be directed against the isotype of the primary antibody.
Polyclonal primary antibodies are generally raised in rabbit, goat, sheep or donkey and are an IgG isotype. The secondary antibody will typically be an anti-IgG H&L (Heavy & Light chains) antibody.
Monoclonal primary antibodies are commonly raised in mouse, rabbit and rat. For example, if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG.
Human immunoglobulin classes, subclasses, types and subtypes:
Other type of reactivities:
The type of conjugation is application dependent.
For enzymatic and biotin detection, e.g. in WB or ELISA, we suggest a secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP) or biotin. Both avidin and streptavidin bind very strongly to biotin and enable signal amplification, regardless of the host species of the antibody.
If a laser light is involved, e.g. in Flow Cytometry, ICC/IF or IHC, we suggest fluorescent detection with a secondary antibody conjugated to a fluorochrome.
We usually recommend using a secondary antibody pre-adsorbed with serum, for western blotting of immunoglobulin-rich tissues and cells. Pre-adsorbed secondary antibodies are less likely to interact with endogenous immunolgobulins and consequently may reduce non-specific background. The secondary antibody should be pre-adsorbed against the same species as the sample on which the detection is performed. For example, a human pre-adsorbed antibody will be required for detection in human tissue.
The advantage of using affinity purified antibodies or IgG fractions will depend on the type of binding expected. Affinity purified antibodies give the lowest amount of non-specific binding whereas IgG fractions contain high affinity antibodies. Indeed, during an affinity purification, high affinity antibodies stay fixed on the matrix and cannot be eluted.
F(ab) and F(ab')2 fragment antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells and B cells) and penetrate tissues more efficiently due to their smaller size. As fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection.
Our secondary antibodies are supplied in different formats: