What's the difference between a direct and indirect assay?
Assays are classed as direct if the label is conjugated directly onto the primary antibody. If the primary antibody is unconjugated, and the label is attached to another molecule, such as a secondary antibody, then the assay is indirect (Figure 1).
Direct and indirect methods have advantages and disadvantages you need to consider when choosing the most appropriate one for your experiment (see Table 1).
Table 1. Advantages and disadvantages of direct and indirect methods.
Suitable to detect high-medium expressed targets as there is no signal amplification generated by the secondary antibody.
Suitable for studying low-expressed proteins because of the signal amplification provided by the secondary antibody.
Time
Conjugated primary antibodies reduce the required number of steps, resulting in a shorter protocol. Only one round of incubation with the sample is necessary because the signal visualization comes from the conjugated primary antibody.
Using a secondary antibody results in a longer protocol due to the additional incubation steps required. In the first step, your sample must be incubated with the unconjugated primary antibody. In the next step, a secondary antibody conjugated to your label of interest will be incubated with the primary antibody to visualize your antigen.
Cost
Conjugated primary antibodies are usually more expensive than secondary antibodies.
Secondary antibodies are relatively inexpensive compared to primary antibodies and can be used to detect different primary antibodies.
Complexity
Fewer steps in the protocol simplify direct methods, meaning experiments are usually easier to design. Dye-antibody combination gives you more flexibility to build a multiplex assay.
You need to select the appropriate secondary antibody or multiple controls, which adds complexity. This is particularly relevant in multiplex experiments, which require several primary antibodies, each targeting different species, and several corresponding secondary antibodies, each conjugated to various dyes.
Species cross-reactivity
Species cross-reactivity is minimized in direct methods as the fluorophore is already conjugated to the primary antibody.
Secondary antibodies may cross-react with species other than the target. The use of pre-adsorbed secondary antibodies can prevent cross-reactivity.
Background
With direct methods, the background is reduced because the primary antibody is already conjugated and less likely to react with other antigens in your sample.
Because you’re adding additional reagents to your experiment, the secondary antibodies may cross-react with other antigens in your sample, resulting in a higher background.
Commonly used labels
Labels used to conjugate antibodies include fluorescent dyes, small molecules (eg, biotin), enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP)), proteins (eg, R-PE, APC, streptavidin), latex or gold nanoparticles, and oligonucleotides. The choice of the label will depend on the experimental application, as shown in Table 2.
Table 2. Labels and their most common applications
Experimental technique
Label(s)
Western blot
HRP, AP, fluorescent dyes
Immunofluorescence
Fluorescent dyes, including Alexa Fluor® dyes
Fluorescent immunohistochemistry (singleplex)
Fluorescent dyes
Fluorescent immunohistochemistry (multiplex)
Fluorescent dyes, oligonucleotides
Chromogenic immunohistochemistry
HRP, AP, Biotin
Flow cytometry
Fluorescent dyes, such as Alexa Fluor® dyes and fluorescent proteins, including Phycoerythrin (PE), Allophycocyanin (APC), and tandem dyes
