Direct vs. Indirect Detection

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Direct vs. Indirect Detection

Immunoassays require antibody binding to the target molecule for detection of protein expression or concentration. In an antibody-based assay, the primary antibody confers specificity, while the label or conjugate attached to the antibody determines the method of detection (fluorescent, enzymatic, etc).


Direct Detection – One Antibody Required – In this method, the primary antibody targeting the molecule of interest is labeled; the labeled antibody binds directly to the target of interest.

Indirect Detection – Two Antibodies Required - In this method, the primary antibody targeting the molecule of interest is unlabeled. Instead, the method of detection is determined by a labeled secondary antibody. This labeled secondary antibody is directed at the constant region of the unlabeled primary antibody.

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The direct method is less sensitive since signal amplification afforded by the secondary antibody is lost

The indirect method is more sensitive since multiple secondary antibodies can bind a single primary antibody


The direct method protocol is shorter due to elimination of the secondary antibody incubation steps

The indirect method protocol is longer due to secondary antibody incubation steps which are eliminated in direct detection


Direct method multiplexing is easier since multiple primary antibodies from the same species can be used together

Indirect method multiplexing is more complicated since secondary antibody can cross react


Non-specific background is lower in the direct method since secondary antibody cross reactivity is eliminated

Non-specific background is higher in the indirect method due to secondary antibody cross reactivity


The direct method is less flexible since the selection of commercially available conjugates can be limited

The indirect method is more flexible because it is possible to use different conjugated secondary antibodies depending on your experiment