How to choose and use antibodies-primary antibody

Choosing a primary antibody

Primary antibodies are antibodies that bind directly to the target protein and have a variable antibody region that recognizes the protein's epitope. The following points should be considered when choosing a primary antibody:

Antibody cloning and methods of manufacture

Clonality depends on whether the antibody is from a different B cell (polyclonal antibody), or from the same B cell from the parent clone (monoclonal antibody). These antibodies have different advantages and limitations.

Broadly speaking, polyclonal antibodies consist of a heterogeneous mixture of antibodies, and each recognising a different epitope of a particular antigen. By binding to a number of different epitopes, polyclonal antibodies can produce a strong signal against the target antigen in relevant applications without favoring a single epitope. However, polyclonal antibodies are in limited supply, vary greatly from batch to batch, and are cross-reactive and lack specificity.

In contrast to polyclonal antibodies, monoclonal antibodies recognise only a single epitope for each antigen. Monoclonal antibodies have high target specificity, low non-specific cross-reactivity and minimal batch-to-batch variation.

The term "recombinant" refers to antibodies produced in vitro using synthetic genes. Compared to conventional monoclonal and polyclonal antibodies, recombinant antibodies provide a long-term, secure supply with minimal lot-to-lot variation. Since the coding sequences of the antibodies are known and defined, they can be further designed and manipulated according to their intended use.

We recommend the use of recombinant monoclonal antibodies when clones suitable for the specific target and application are available to ensure reproducible experiments and a long-term supply of antibodies. For applications where polyclonal antibodies have traditionally been used (e.g. analysis of low abundance targets or simultaneous detection of multiple post-translational modifications), recombinant polyclonal antibodies can provide an ideal solution. Recombinant polyclonal antibodies are mixtures of carefully selected individual recombinant monoclonal antibodies designed to recognise different epitopes on the same antigen. As a result, they can provide excellent sensitivity, specificity and re-producibility that only recombinant antibodies can offer.

Antibody specificity confirmed by knock-out validation

Good antibodies are target specific and recognise proteins of interest even at low expression levels. However, many studies have shown that not all antibodies have this specificity and many cross-react with non-target proteins.

Knockout (KO) validation is one of the most recognized and reliable validation methods for antibody specificity. This robust technique confirms antibody specificity by testing in KO cell lines, cell lysates or tissues that do not express the target protein. The specific antibody should produce no signal in the KO cell line, but a specific signal in the wild-type cell line. In this way, the KO validation becomes a true negative control.

Figure 14 below shows an example of KO validation of a KI-67 antibody in immunocytochemistry (ICC), with KI-67 knockout HAP1 cells (bottom) showing no Ki67 (green) expression.

Figure 14. Immunocytochemistry/immunofluorescence images of Ki67 antibody in wild-type cells (top) and Ki67 knockdown HAP1 cells (bottom). Anti-Ki67 (ab92742) with goat anti-rabbit IgG (Alexa Fluor® 488) in green, anti-α-microtubulin (Alexa Fluor® 594) in red and nuclear DNA labelled with DAPI in blue.

We recommend that you select antibodies that have been validated for multiple applications, preferably using KO technology. Alternatively you can self-validate the antibody using appropriate KO cell lines, KO cell lysates or tissues.

Immunogen details

The discovery of antibodies usually begins with immunisation of the host animal with immunogens. These immunogens can be full-length proteins, peptides, or whole cells.

The immunogen used will determine which region of the protein the antibody binds to. For example if you want to detect a cell surface protein on a live cell by FACS, you should choose an antibody that targets the extracellular structural domain of that protein.

Sample processing

An antibody is specific for an epitope in a particular conformation. Since sample processing changes the conformation of the epitope (e.g. fixation leads to protein cross-linking by formaldehyde-induced methylene bridges), some antibodies will only work on samples processed in a certain way. Many antibodies will only recognise proteins that have been reduced and denatured, as this exposes epitopes that would otherwise be hidden. On the other hand, some antibodies only recognise epitopes on proteins in their native state.

For immunohistochemistry, certain antibodies are suitable for unfixed frozen tissue, while others require an antigen retrieval step to expose the epitope if they have been formalin-fixed and paraffin-embedded. We suggest verifying if the antibody datasheet specifies any limitations regarding sample processing.

Host species

For indirect detection using secondary antibodies, ideally choose a primary antibody that has been raised in a different species to your sample. This prevents cross-reactivity of the secondary (anti-immunoglobulin) antibody with endogenous immunoglobulins in the sample. For example, if you are studying a mouse protein, choose a primary antibody raised in a species other than a mouse, such as a rabbit. Since cross-reactivity arises due to the existence of host antibodies in the sample, it poses a challenge for tissue samples but not for cell lines.

Suppose you have to use a primary antibody that has the same host species as the tissue sample. In this case, you will need to carefully consider how to modify the protocol to reduce background staining. Alternatively, to avoid cross-reactivity, you can use a chimeric antibody consisting of structural domains from different species.

You do not need to worry about the host species of the primary antibody when using cell lysates that do not contain any endogenous immunoglobulin (IgG) for applications such as Western blotting or direct detection experiments using primary antibody conjugates.

Carriers and preservatives

Typically antibodies are stored in phosphate buffered saline (PBS) solutions containing carrier proteins such as bovine serum albumin (BSA) and preservatives such as glycerol and sodium azide. While these are important components for maintaining antibody stability and preventing contamination, they can prevent efficient attachment of labels (e.g., fluorescent dyes, enzymes, and metals), affect live cell systems, and may even interfere with highly specialised hardware setups.

In a typical conjugation reaction, BSA competes with the primary antibody for adsorption to the label of interest, thereby greatly reducing conjugation efficiency. Sodium azide in the antibody solution can be toxic to cells, thus limiting the use of antibodies in cell culture and negatively affecting conjugation. Therefore, if you intend to conjugate a primary antibody or stain live cells with a primary antibody, we recommend that you choose an antibody formulation that does not contain carriers or preservatives.

References

1.Weber et al. (2017) From rabbit antibody repertoires to rabbit monoclonal antibodies. Experimental and Molecular Medicine. Vol. 49: e305

 2.Rajewski, Klaus (1996) Clonal selection and learning in the antibody system. Nature. Vol. 381: 751-758.

3.Cheung et al. (2012) A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nature Biotechnology. Vol. 10(5): 447-454

4.Ascoli and Aggeler (2018) Overlooked benefits of using polyclonal antibodies. BioTechniques 65(3): 127-136