A secondary antibody is a special kind of antibody used in experiments to help detect or measure specific proteins or other molecules. It works by attaching to a primary antibody, which is already bound to the target (called the analyte).
Secondary antibodies are used in many types of scientific tests and are available in lots of different forms. One big benefit of using them is that they can amplify the signal, which helps scientists see or measure small amounts of the target more easily.
If you’re planning an experiment or setting up an assay, it’s easy to buy secondary antibodies that match your specific needs—whether you need enzyme-linked, fluorescent, or biotin-labeled options.
Why Use Secondary Antibodies?
Secondary antibodies are used in a method called indirect detection. Here’s how it works:
- First, a primary antibody finds and sticks to the target molecule.
- Then, a secondary antibody sticks to the primary antibody.
- The secondary antibody is usually labeled with something that can be seen or measured—like an enzyme or a glowing fluorescent tag.
To make a secondary antibody, scientists inject an animal (like a goat) with antibodies from another species (like a rabbit). The animal’s immune system creates antibodies against those, which are then collected and purified.
Since more than one secondary antibody can attach to each primary antibody, the signal becomes stronger, making it easier to detect targets that are present in very small amounts.
How Specific Are Secondary Antibodies?
Secondary antibodies can be made to recognize many types of primary antibodies—or just very specific ones—depending on how they’re designed.
Antibody Classes and Subtypes
Often, secondary antibodies are made to detect a mix of all antibody types from a particular species (like all mouse or rabbit antibodies). These include:
- Different classes: IgA, IgD, IgE, IgG, and IgM
- Different subtypes: like IgG1, IgG2, IgG3, IgG4, etc.
- Different chains: both heavy chains (H) and light chains (L)
This broad approach makes secondary antibodies very versatile—they can work with most primary antibodies from the same species.
But sometimes, scientists need more precision. In those cases, secondary antibodies can be made to recognize only:
- One specific class or subtype
- Just one kind of light chain (either kappa (κ) or lambda (λ))
This added specificity is especially useful when you’re running experiments with multiple antibodies from the same species.
By choosing secondary antibodies that only bind certain types, you can combine them in the same experiment without mixing up the signals.
Preventing Unwanted Binding
Sometimes, secondary antibodies can accidentally stick to the wrong targets, which can lead to confusing or incorrect results in experiments. To help prevent this, companies use two special processes: affinity purification and cross-adsorption.
What is Affinity Purification?
Affinity purification is a method used to isolate only the useful secondary antibodies. It works like this:
- Scientists attach the target antibody (like mouse IgG1) to a solid surface.
- Then they pass a mixture of antibodies (from, say, a goat) over it.
- Only the goat antibodies that specifically stick to mouse IgG1 are kept—these are the desired secondary antibodies.
What is Cross-Adsorption?
After purification, the antibodies can go through cross-adsorption, which removes any that still might stick to the wrong things, like:
- Antibodies from a different species
- Other classes or subtypes of antibodies
This step is especially important in:
- Sandwich ELISA: where the detection antibody should not stick to the capture antibody
- Immunohistochemistry (IHC): where the secondary antibody should not bind to proteins in the tissue being studied
Using cross-adsorbed secondary antibodies helps ensure that results are accurate and specific.
Secondary Antibody Fragmentation
In the past, secondary antibodies were usually used in their full (whole) form, like full-length IgG antibodies.
But now, scientists also use antibody fragments, such as Fab and F(ab’)₂. These are smaller pieces of the antibody that still work for detection.
Why Use Antibody Fragments?
These fragments do not have a part called the Fc region. That’s important because:
The Fc region can sometimes stick to immune cells (like macrophages, dendritic cells, or B cells), even if it’s not supposed to.
This unwanted binding can cause non-specific background signals in experiments.
By removing the Fc region, antibody fragments avoid this problem, leading to cleaner, more accurate results.
Also, because they’re smaller, these antibody fragments can:
- Enter tissues and cells more easily
- Move around more quickly than whole antibodies
This makes them especially useful in experiments where access to deep or tightly packed tissues is important.
Uses of Labeled Secondary Antibodies
Secondary antibodies are often attached to different labels that help detect or measure the presence of specific proteins. The type of label you choose depends on:
- The test you’re doing (e.g. Western blot, ELISA, IHC, etc.)
- Whether you need to measure multiple things at once (this is called multiplexing)
1. Enzyme Labels
Many secondary antibodies are labeled with enzymes like HRP (horseradish peroxidase). These are commonly used in:
- ELISA (to produce a color change with TMB)
- IHC (to stain tissues using DAB)
- Western blots, where HRP reacts with special chemicals (ECL) to produce light—this method is very sensitive and can detect even tiny amounts of protein.
2. Fluorescent Labels
Other secondary antibodies are tagged with fluorescent dyes or fluorescent proteins, such as:
- Alexa Fluor®, Cyanine dyes, or R-phycoerythrin: These are used in experiments like immunocytochemistry (ICC) or flow cytometry
- Fluorescent tags are especially useful when you want to detect multiple proteins at the same time (multiplexing), but you need to plan your experiment carefully so the signals don’t overlap.
- Some fluorescent tags are “tandem dyes” (e.g., PE/CY5), which let you detect more than one signal using the same light source—handy for flow cytometry.
3. Biotin Labels
Secondary antibodies can also be labeled with biotin, a small molecule that sticks very tightly to proteins like avidin or streptavidin.
The strong interaction is useful in techniques like the ABC method, which is used in IHC to boost the signal and make the target protein easier to detect.