When using immunochemical methods, we place great emphasis on the selection of the primary antibody. We study leaflets and pictures in them. We check whether the given primary antibody is suitable for the application and the target organism. We are looking for whether it has already been used in the publication. But what about the secondary antibody? Do we always pay the same attention to choosing a secondary antibody?

It is for this reason that we have prepared this material for you in order to summarize the aspects suitable to consider when choosing a secondary antibody. We believe that some of the above tips will help you orient yourself in the topic.

What is a secondary antibody:
A secondary antibody is an antibody that does not bind directly to the antigen of interest, but to the primary antibody. The primary antibody conjugated to the label can be used directly to detect antigens, however, the use of secondary antibodies offers a number of advantages. E.g. the specificity of the secondary antibody to the regions of the primary antibody allows more secondary antibodies to bind to the primary antibody, thereby amplifying the signal and increasing the sensitivity of the method. An undeniable advantage is the ability to conjugate the secondary antibody to a variety of colored labels or enzymes. Conjugation of antibodies with a detection molecule gives us considerable flexibility and variety of experiments and allows the detection, sorting and purification of target proteins. The use of universal secondary antibodies is also a more economical option. Not only is the production of labeled primary antibodies relatively expensive. In addition, the presence of the label itself may cause the antibody as a malfunction.

How is the functionality of the secondary antibody characterized?
• An important parameter of a secondary antibody is the specificity or degree of detection of the primary antibody.
• Sensitivity refers to the amount of antigen that can be detected by an antibody. Sensitivity depends not only on binding affinity but also on antibody labeling.
• Antibody consistency refers to the ability of an antibody to achieve repeatable results using different lots. The source of the antibody and its clonality (polyclonal vs. monoclonal) have a direct effect on consistency.
Antibody structure
The antibody is a Y-shaped protein. It consists of 4 polypeptide chains – 2 identical heavy chains (H, heavy) and 2 identical light chains (L, light) (Fig. 1). Both heavy chains are connected to each other in the hinge region. At this point, the antibody can be cleaved by papain into Fc and Fab portions. The crystallizable region Fc (Fragment crystallizable) is a region important for the function of the antibody in the immune response. Within the individual antibodies, the Fc region does not differ much. Fab (antigen-binding fragment) regions play a role in the binding of the antigen itself. If we take a closer look at the Fab region, we can see that the antibody at this site is made up of a variable (V, variable) and a constant (C, constant) domain. It is the variable regions that are the parts of the chains that differentiate the individual antibodies and are responsible for the specific recognition of the antigen.

Secondary antibody selection
The preparation of secondary antibodies is performed by immunizing an animal with another antibody (from a different animal species). Thus, the specificity of the secondary antibody produced is determined by the nature of the immunizing antigen, i.e., the specificity of the secondary antibody to the animal organism, antibody subclass, and fragment is determined in this way.

1. Host organism and reactivity
The first step in selecting a secondary antibody is to select a host organism. We need to find an antibody that will react with our primary antibody; or with the animal organism in which the primary antibody was prepared. E.g. if we have a primary antibody prepared in a mouse, the secondary antibody must be directed against the mouse (anti-mouse). The host organism is then the animal in which the secondary antibody was prepared. Using our example, the secondary antibody here must be made from an animal other than a mouse, such as a goat (Fig. 2).

The most commonly used are secondary antibodies against anti-mouse, anti-rabbit, rat (anti-rat), hamster (anti-hamser) and guinea pig (anti-Guinea Pig). This is the case when using polyclonal primary antibodies. If a primary monoclonal antibody is used, the secondary antibody will most often be targeted to the mouse (anti-mouse).

It should also be borne in mind that the host organism of the primary antibody differs from the type of sample we are working with. (You can read about possible mouse-on-mouse detection in the next article.)

2. Selection of conjugated detection molecule
For immunochemical methods, antibodies are usually conjugated to a detection molecule. The selection of a particular conjugate depends directly on the method used (Fig. 3).

For ELISA and western blot (WB), the most popular variants are enzymatically labeled secondary antibodies, most commonly horseradish peroxidase (HRP) and alcalic phosphatase (AP). HRP is a more economical and stable variant compared to AP. Due to its properties, it has become popular especially in chemiluminescent detection. However, AP shows increased sensitivity, so it found its place more often in colorimetric detections. Another alternative to enzymatic labeling is biotin labeling. The ability of avidin and streptavidin to bind to biotin and form a complex allows up to four-fold amplification of the signal, independent of the host species of the secondary antibody (Read more about the method in the article on the use of ABC complex for IHC).

Fluorescent molecule-labeled secondary antibodies, such as Alexa Fluor, DyLight, FITC, TRITC and others, are used for flow cytometry and immunofluorescence staining (IF) (A list of abbreviations is given at the end of the article.). When selecting a fluorescent label, the excitation and emission spectra of the fluorochrome for the experiment should be considered. Fluorescent labels are especially popular in the immunofluorescence detection of several antigens simultaneously. An appropriate combination of secondary antibodies is required to avoid overlap between channels (Fig. 4). For these purposes, it is appropriate to use fluorescent antibodies with a narrow emission spectrum. The use of a suitable recording device (microscope, scanner, etc.) is a matter of course.

Fluorescent antibodies are also used in the WB method. Compared to chemiluminescent detection of WB on the enzyme / substrate principle, where the signal strength can be significantly affected by the kinetics of the ongoing reaction, the main advantage of fluorescently labeled IRDye antibodies is the possibility of detecting more than one protein. The signal here is directly proportional to the amount of target protein, which allows for easy quantification. Another positive aspect of the use of IRDye antibodies in the detection of antigens in WB is the stability of the formed complex, the possibility of storing the membrane and its re-imaging. Unfortunately, we must not forget the higher acquisition costs of labeled antibodies and, last but not least, the cost of instrumentation.

The last type of conjugate label mentioned here is the association of an antibody with gold nanoparticles of different sizes. An example of such an application is electron microscopy.

3. Class and subclass of antibodies
Based on the structure, antibodies are divided into individual classes and subclasses. The variation in the Fc region of the heavy chain fragment divides the antibodies into the following classes: IgA (1-2), IgD, IgE, IgG (1-4) and IgM (Table 1). The heavy strings are then denoted by the corresponding letter of the Greek alphabet: α, δ, ε, μ and ϒ. IgG and IgA isotypes can be further divided into subclasses according to differences in the heavy chain. As with heavy chains, light chains are divided into lambda (λ) and kappa (κ). Only one type of string can be present at a time – λ or κ.

Isotype or class: IgG (ϒ heavy chain), IgM (μ) IgA (α), IgE (ε), IgD (δ)
Subclass: IgG1 (ϒ1 heavy chain), IgG2 (ϒ2), IgG3 (ϒ3), IgG4 (ϒ4), IgA1 (α 1), IgA2 (α2)
Types: λ and κ light chain
Tab. 1 .: Human immunoglobulins – isotypes, subclasses and types

Secondary antibodies must be directed against the primary antibody isotype. E.g. for the mouse primary IgM antibody, we will choose a secondary anti-mouse IgM-detecting antibody.

Another basic question during secondary antibody selection is: Is your primary antibody polyclonal or monoclonal? Polyclonal antibodies contain a mixture of several immunoglobulin G isotypes (eg, IgG1, IgG2a). Therefore, to improve the detection of the target protein, it is best to use a secondary antibody that distinguishes all isotypes, or IgG H + L secondary antibodies.

In contrast, monoclonal primary antibodies contain only one immunoglobulin isotype. Therefore, it is important to use a secondary antibody that specifically recognizes a given isotype. This information is usually provided in the primary antibody package insert.

It follows from the above that the most common primary and secondary antibodies are produced in the IgG variant. We meet other types rather occasionally. Antibodies specific for each subclass can be used to distinguish between primary antibodies by multiple staining.

4. Preabsorbed antibodies
Preabsorption of an antibody is an additional purification step to increase the specificity of the antibody. The principle of purification is to flow the secondary antibody through a column that contains a matrix with immobilized proteins from the serum of potential animal species that could cross-react with the antibody. Non-specific antibodies are captured on the column, while highly specific secondary antibodies flow freely. The degree of cross-reactivity is then monitored by ELISA or WB. It usually reaches 1%. In the case of selection of preabsorbed antibodies, we select such that they are absorbed by the animal species we detect. E.g. when studying human tissue samples, we select a secondary antibody that will not recognize human proteins because this type of binding causes false positive results or a high background.

The use of preabsorbed antibodies is recommended for immunohistochemical staining of a sample with a high proportion of endogenous immunoglobulins. For these experiments, we use a secondary antibody preabsorbed against the same species as the sample. By using preabsorbed antibodies, the cross-interaction between the secondary antibody and endogenous IgG is reduced and the non-specific background is reduced.

To reduce the non-specific interaction, we can also block the background with sera from the same species as the secondary antibody used.

5. Affinity purified antibodies or IgG fractions
Another decision criterion is the degree of purity of the antibody. In the experiments we have the possibility to use the complete IgG fraction or affinity purified antibodies.

Unpurified IgG fractions containing the whole immunoglobulin complex. The entire fraction is usually obtained by isolation with Protein A. The antibodies can then be affinity purified to remove antibody subclasses or antigen-non-specific antibodies from the sample. This process is performed by separating specific antibodies from other antiserum proteins and non-specific immunoglobulins by solid phase affinity chromatography.

The advantages of affinity purified antibodies are increased specificity, decreased non-specific background interaction, increased sensitivity, and higher batch-to-batch consistency. However, it should be borne in mind that high affinity antibodies may be eliminated during purification processes due to their affinity for the matrix. For this reason, the use of an IgG fraction is rather recommended for the detection of low concentration target proteins. The choice between the IgG fraction and the purified antibodies is based on our expectations. Affinity purified antibodies have fewer non-specific interactions, while IgG fractions contain high affinity antibodies. In general, IgG fractions are suitable for turbidimetric assays, while most immunochemical methods should use affinity purified antibodies.

6. Fragment antibody format – F (ab) or F (ab´) 2
The format of the secondary antibody used depends on its structure itself. The use of complete IgG is common, but in special cases it is appropriate to use antibody fragments. Antibody fragments are made from complete IgG by papain cleavage in the hinge region. In this way, individual regions F (ab) are created (Fig.5.). The second variant is fragments F (ab´) 2. These are also formed by proteolytic cleavage of IgG, but using pepsin. In both cases, most of the Fc region is deleted.

Both types of antibodies are smaller than the complete IgG molecule, thus allowing better penetration into the tissue and thus better antigen recognition and increasing the signal during IHC staining. A second advantage of fragment antibodies is the absence of their Fc fragment. Due to the absence of a crystallization region, non-specific binding between the Fc regions of antibodies and Fc receptors of cells is eliminated. Therefore, fragment antibodies are particularly popular in the labeling of cells with high levels of surface Fc receptors – or in the labeling of macrophages, dendritic cells, neutrophils, NK cells and B-lymphocytes and in the detection of antigens in tissues with high levels of endogenous Fc receptors – spleen and thymus.

F (ab) antibodies are further used to block endogenous immunoglobulins in cells and tissues. After classical blocking of the tissue with serum in the IHC method, the sample is incubated with F (ab) fragments. This blocks endogenous immunoglobulins and avoids the increased background that would occur when classical secondary antibodies bind to endogenous immunoglobulins.

In addition, F (ab) can be a powerful tool in multiple staining using primary antibodies of the same species. Because the F (ab) fragment secondary antibodies contain only one antigen binding site (are monovalent), they cannot bind any other molecule in the case of sequential staining (Fig. 6). F (ab´) 2 secondary antibodies are not suitable for this purpose due to the 2 binding sites. They could bind a second secondary antibody in sequential staining and show false positive results.

What things to keep in mind when choosing a secondary antibody? Here is a summary of the questions you should ask yourself when choosing a secondary antibody.

1. In which animal organism was your primary antibody produced?
2. Which conjugate detection molecule is suitable for your application?
3. Do you use an enzymatic detection method? Do you prefer HRP or AP?
4. Do you use a fluorescence detection method? Take a look at the range of emission spectra, especially if you do multiple staining.
5. Will you use the biotin technique?
6. What is the class / subtype or subclass of your primary antibody?
7. Do you need an affinity purified antibody or IgG fraction?
8. Do you need a preabsorbed secondary antibody for your purposes?
9. Do you need an F (ab) or F (ab´) 2 fragment antibody for your experiment?