Antibody Standard Validation

The success of any immunodetection experiment depends on the quality of the antibodies which are employed. However, antibody reagents vary significantly and when selecting an antibody for a downstream application it is a good idea to spend some time ensuring that not only has it been tested in the chosen experimental setup but that it also demonstrates the required specificity, sensitivity and reproducibility. This valuable data is generated during the antibody manufacturing and validation process and can be found on the product datasheet with which the antibody is supplied.

Antibody Information

Detail regarding the nature of the immunogen used to generate an antibody can provide a good indication as to whether the antibody will work effectively in the chosen application. Immunogens are typically full-length proteins, protein fragments or peptides, although cells and whole organisms may also be used. As an example, if a researcher wishes to use immunodetection to confirm expression of a C-terminal recombinant protein fragment within a transfected cell line, there would be little point in choosing an antibody which was raised against the N-terminus of the target protein. Or if the aim is to immunodetect a cell surface protein utilizing a protocol which avoids lysis or cell permeabilization, it would be wise to select an antibody raised against an extracellular rather than an intracellular region of the protein.

The clonality of the antibody should also be considered. Polyclonal antibody preparations, produced by animal immunization, consist of a pool of antibodies of which only a proportion recognize the target of interest. These reagents can vary considerably in performance from one immunized animal to another, meaning that a permanent antibody supply with consistent and reproducible binding capabilities cannot be guaranteed. Monoclonal antibodies, produced via hybridoma technology, recognize a single antigenic epitope and can easily be produced in large quantities. Since they demonstrate uniform performance they are often preferred, particularly for long-term projects such as screening campaigns.

While immunogen affinity purification can be used to enrich the target-specific antibodies within a polyclonal antibody preparation, not all polyclonals are purified in this manner. Many are instead subjected to class-specific affinity purification, which relies on the differential binding capacity of antibody Fc regions to immobilized capture proteins such as Protein A or Protein G. Although this method of purification enriches certain antibody classes and results in the removal of most serum proteins, it does not eliminate non-specific antibodies, meaning that some cross-reactivity may remain. Monoclonal antibodies do not require immunogen affinity purification since the antibody of interest is the only antibody within the preparation.

It can often be helpful to know the isotype of the antibody which is to be used for immunodetection, since different isotypes vary in terms of biological properties and functions, which may influence the level of background staining that is produced. Isotype controls share the same isotype as the antibody which is used in the downstream application but lack any specificity for the target antigen. They are often used to distinguish background staining from the specific antibody readout.

Although not often performed routinely by antibody manufacturers, epitope mapping can provide an additional level of information about an antibody’s binding properties. Antibodies raised against a protein in its native conformation commonly bind to a conformational or discontinuous epitope, and if these antibodies are used for immunodetection of a reduced and denatured form of the protein it is highly unlikely for binding to be successful. Likewise, an antibody raised against a peptide immunogen may not be able to recognize and bind its epitope when the protein is in the fully folded state.

Target Information

By studying the background information surrounding the target protein, researchers can gain a clear understanding of the likelihood of detecting it in the chosen sample material. Our Prestige Antibodies^® are supported by the data of the Human Protein Atlas, which provides a wealth of information regarding protein expression at the tissue level and within cells, in addition to detail regarding differences in expression between normal and cancer tissue. This powerful resource can be used freely to explore expected patterns of tissue or cellular localization, as well as for the selection of antibody reagents and to choose appropriate positive and negative control materials for a study.

Another useful source of information regarding a molecular target is UniProt which can, for example, be used as a point of reference regarding the theoretical molecular weight of a protein. Detail about the number of different isoforms which are known to exist, whether specific isoforms are more abundant under certain conditions or within particular cell or tissue types, and how the protein is post-translationally modified can also be found. It is worth noting that post-translational modifications can have a significant impact on the theoretical molecular weight of a protein, leading to Western blot detection of bands which may be considerably larger than originally anticipated.

In some cases, it may be necessary to enhance expression of the target protein to immunodetectable levels using defined experimental conditions. For example, hypoxia may be used to promote expression of HIF proteins, autophagy induction is often required to generate measurable levels of proteins such as LC3, and the production of nuclear or mitochondrial fractions can be used to facilitate detection of proteins specific to those cellular sub-compartments. It is often worth consulting the literature to discover which techniques other research groups have employed to identify specific protein targets, especially those that are more challenging to detect.

Species Validation

In many cases, an antibody which has been raised against a human antigen will also recognize that same antigen in samples derived from other species such as mouse, rat or rabbit, due to a high degree of cross-species sequence homology. This cannot however always be assumed as a given, and details of the species against which the antibody has been tested and found to work will be listed on the product datasheet. Often, species against which the antibody has been tested and found to be unsuccessful will also be detailed, along with predicted reactivities against untested species.

To estimate the predicted reactivity, free online resources such as MultAlin and ClustalW2 are excellent tools for performing sequence alignments. These can provide information regarding the percent homology between forms of the same protein from different species and can also be used as a quick visual aid to determine where the greatest degree of similarity or dissimilarity lies.

Application Validation

Product datasheets will typically list all the applications in which an antibody has been tested, with the most common examples of these being Western blot, immunocytochemistry, immunohistochemistry (IHC), ELISA and flow cytometry. An important point to note with regards to IHC is that an antibody may behave very differently when it is used to probe frozen tissues than when it is employed against formalin-fixed paraffin-embedded (FFPE) samples. It cannot be assumed that successful antigen binding within frozen tissue is indicative of a similar outcome in FFPE samples, and vice versa, and the datasheet should clearly specify the sample type against which the antibody has been validated for IHC.

For applications in which the antibody is listed as being suitable for use, recommended protocols are often provided for guidance. These usually include suggested antibody starting dilutions and incubation times, although every experimental set up will always require the end user to carry out optimization.

An antibody demonstrating good performance in a Western blot should detect a clean band of the expected molecular weight, with minimal background staining, while a high performing antibody for immunocytochemistry or IHC should demonstrate the anticipated pattern of staining as defined in the literature. Where an antibody is described as suitable for ELISA, it is helpful to see additional detail regarding the ELISA format in which it was tested and, if a sandwich ELISA was performed, whether a specific paired antibody has been recommended. Antibodies for flow cytometry are often provided directly conjugated to fluorescent dyes to aid detection. For these it is beneficial to see the excitation and emission wavelengths of the attached dye, so that cross-talk can be avoided when setting up a multi-color flow cytometry experiment.

Storage and Handling Information

While some antibodies are provided as solutions, which can include ascites fluid, tissue culture supernatant or a purified antibody product in a defined buffer, others are supplied in a lyophilized format requiring reconstitution. Liquid antibodies should always be centrifuged briefly before being opened, to prevent loss or contamination of the product. Lyophilized antibodies should be carefully reconstituted according to the manufacturer’s instructions. Since antibody lyophilization often occurs in the presence of various buffer components, it is essential that reconstitution instructions are followed precisely to avoid inadvertently changing the final buffer composition and impacting on antibody performance.

Many antibodies exhibit a loss of performance upon freezing, or because of repeated freeze-thaw cycles. The antibody datasheet will detail appropriate storage conditions on a product-specific basis. Antibodies which have been conjugated to fluorescent dyes or fluorescent proteins cannot usually tolerate freezing and may have a shorter shelf-life than unconjugated antibody products. They are typically provided in opaque tubes to avoid light exposure, and when working with fluorescently-conjugated antibodies it is essential that storage and any downstream incubation steps should also take place in the dark to prevent the fluorescent signal from being lost.

Lot Validation

Each unique lot of an antibody should be supplied with lot-specific data including the concentration of that lot and validation data for each of the applications in which it has been tested. It should never be assumed, especially in the case of polyclonal antibodies, that every batch of an antibody will show the same pattern of behavior, and it is good practice to test each new lot in-house within the intended application against appropriate controls and alongside the previous antibody lot before using it with precious sample materials.

Citations

Antibody product pages include a list of publications in which the antibody has been cited for use. PubMed and CiteAb are excellent sources of information regarding suggested immunostaining protocols and usually provide detail regarding sample types against which the antibody has been used successfully. In the case of monoclonal antibodies, a quick online search for the clone name can often provide additional information such as the originating source and a more in-depth description of the method of production.

Quality Control

We are a global company with manufacturing facilities worldwide. Our development and manufacturing processes are subject to rigorous quality control and quality assurance measures, and each of our antibody products is supplied with a comprehensive Certificate of Analysis and Product Information Sheet.

For Research Use Only. Not for use in diagnostic procedures.

Unless otherwise stated in the Product(s) specifications, any Antibody product is sold for internal research use only and may not be used for any other purpose, which includes but is not limited to, any commercial, diagnostic, or therapeutic use. Our validation processes pertain only to research uses and do not confirm or assure that our antibodies can be used for any unauthorized uses as set forth herein.