by Woo Mun Wah and Will Olds
Antibodies are among the most commonly used reagents in academic and industrial biotechnology research. It is the quintessential tool used in research applications such as western blotting, immunofluorescence, flow cytometry, immunohistochemistry, and immunoprecipitation. The boom in both antibody companies and number of antibodies available on the market has made antibody selection an increasingly difficult task since not all antibodies are made to the same quality standards or are suitable for all applications.
With increasing pressure to produce results under limited time and resources, bioentrepreneurs and researchers can benefit from learning how to cut through the noise and find a suitable antibody for their experimental needs. In this article, we share what is involved in making good quality antibodies and how to identify a good antibody company to help researchers streamline the process of antibody selection.
Purchasing antibodies: the good, the bad, and the ugly
The truth behind antibody manufacturing is that not all antibodies are made and marketed directly to consumers. Instead, it is all too common for many antibody companies to source their products from various third-party contract manufacturers, who in turn supply multiple antibody companies. On top of that, companies routinely buy and sell products between themselves, resulting in the exact same antibody being marketed under different brands (Figure 1).
Figure 1. Sourcing of antibodies from multiple manufacturers and re-selling between antibody companies may lead to consumers unknowingly purchasing identical products, albeit under different brands. This process has arisen from a lack of regulation regarding antibody supply chain.
As a result, scientists may find themselves testing the same product, albeit from different companies, with no change in technical performance. One way to tell if this is the case is to pay attention to validation images supplied with the product; if the validation images look the same, chances are that this antibody was sourced from the same manufacturer.
A corollary of this practice is that critical information such as validation data, protocols for use in various applications, and antigen information may be lost when antibodies are resold and rebranded. This may result in unnecessary frustration and time spent on the end-user’s part when optimising and troubleshooting experiments. Ideally, a good antibody company should provide reliable, detailed records of how antibodies are made and validated, regardless of whether they are manufactured by the company itself or externally sourced. In addition, companies should have policies in place to guarantee consistency in the quality of their product – for instance, an exchange policy for antibodies found to come from a defective batch.
Ideally, a good antibody company should provide reliable, detailed records of how antibodies are made and validated, regardless of whether they are manufactured by the company itself or externally sourced. In addition, companies should have policies in place to guarantee consistency in the quality of their product – for instance, an exchange policy for antibodies found to come from a defective batch.
Increase your antibody success in 5 moves
So, how can researchers select suitable antibodies for their research needs?
1. Know your immunogen and antigen
How much do you understand about your protein of interest? It is critical to know if the protein of interest has multiple localizations, isoforms, cleavage sites or post-translational modifications. These factors are often represented in the amino acid sequence or can be easily looked up in databases such as Uniprot or the Human Protein Atlas.
The common industry practice among most antibody manufacturers is to design and manufacture antibodies based on a short peptide of approximately 10 to 20 amino acids (aa), which corresponds to a specific region of the full-length protein. However, given that the average length of eukaryotic proteins is 472 aa , the peptide used would not have been a good representative of the native protein.
Therefore, it is a good practice to check the sequence of your protein of interest against the immunogen to ensure that the antibody fits the purpose of your study (see Table 1). For example, the Proteintech Caspase 9/P35/P10 Antibody (10380-1-AP), which uses a fusion protein antigen that spans from 141-392 aa encoded by mRNA transcript BC002452, would be able to recognize both p35 and p10 subunits after cleavage. It would not be possible for an antibody to recognize both subunits if a specific peptide sequence was used instead, and an antibody made that way would most probably recognize either only the p35 or p10 subunit.
Table 1: Differences between peptide and protein immunogens
Full Length Protein
Good for generating antibodies that will detect a single isoform of a protein, for instance post-translational modification such as phosphorylation.
Good for generating antibodies which recognize multiple regions of the protein which is better for multiple applications, and for studying protein modifications over time e.g processing of proprotein.
|Non-specific or have low affinity, as peptides do not fold the same way as full-length proteins||
May have reactivity across different species.
2. The different types of antibodies: polyclonal & monoclonal antibodies
Polyclonal antibodies are a mixture of heterogeneous antibodies produced by injecting an immunogen into an animal. These antibodies can recognize and bind to many different epitopes of a single antigen (Figure 2A). By contrast, monoclonal antibodies are made using identical immune cells descended from a single parental cell. This means that the monoclonal antibodies recognize just one epitope of an antigen (Figure 2B). The advantages and disadvantages of using either type of antibodies are listed in Table 2.
Figure 2. A) Polyclonal antibodies bind to the same antigen, but multiple epitopes whilst B) monoclonal antibodies bind to the same epitope on a target antigen.
Table 2: Pros and cons of using polyclonal and monoclonal antibodies.
Versatility and robustness for multiple applications
The ability to recognize multiple epitopes results in higher affinity and sensitivity for native proteins, making polyclonals favourable for target capture in ELISAs, immunoprecipitation, immunohistochemistry and immunofluorescence.
Quick and cost-effective to produce
Since polyclonal antibodies are produced directly from animals, they can be produced relatively quickly (+/- 3 months) and less expensively than monoclonals.
Homogeneity and batch-to-batch reproducibility
Identical antibodies can be produced in large quantities, making monoclonals favourable for diagnostic and therapeutic applications that require defined components/precise mechanisms of action.
Low cross-reactivity and background noise
Ability to bind to a single epitope makes monoclonals more suited for target detection in quantification assays.
Mixture of antibodies differs between batches
Although many antibody companies have developed efficient ways to produce highly consistent batches and mitigate cross-reactivity with additional validation and affinity purification, the final product is still less well defined than monoclonals which makes them unsuitable for certain therapeutic uses.
More costly and time-consuming to develop
The need to generate hybridized clones, which can take up to 6 months, naturally drives up the cost of production.
3. Method of purification
Two methods are generally used for antibody purification: affinity purification and protein A purification. The former relies on antibody-antigen interactions for purification, which specifically selects for antibodies that have specific binding affinity to the protein of interest. This process usually yields a lower concentration of antibodies, but the end product contains a higher percentage of antibodies which are target-specific as unrelated antibodies have been removed.
Protein A purification on the other hand purifies any IgG antibodies based on antibody-protein A interactions, which selects for any IgGs available in the serum regardless of whether they bind the target antigen or not. Thus, this process yields a higher concentration of antibodies, but increases the chance of getting nonspecific antibodies. Information regarding the method of purification can be found in the documentation accompanying each antibody.
4. Use antibody search engines
Upgrade your search by using specialised antibody search engines instead of generic search engines. Antibody search engines offer convenience and save time by comparing antibodies across most antibody manufacturers and companies on a single platform. Crucially, they also often provide useful validation data and user reviews up front so you can make an informed decision. Examples of antibody search engines include Antibodypedia, CiteAb, AntibodyResource and Biocompare.
5. Identify a responsible antibody company
Earlier in the article, we covered the practices that define a good antibody company. Here are some helpful questions to identify a reliable one:
- Does the antibody company manufacture their own antibodies, or source from other manufacturers/companies?
- Does the antibody company have a stringent manufacturing and validation process?
- Does the antibody company have an open data access policy where validation data, documentation, protocols and immunogen sequences are easily available?
- Does the company have sales terms and conditions that are customer-centric?
- Is there local support available, such as technical support, logistics and inventory?
1 Tiessen, A. et al. (2012) Mathematical modeling and comparison of protein size distribution in different plant, animal, fungal and microbial species reveals a negative correlation between protein size and protein number, thus providing insight into the evolution of proteomes. BMC Res. Notes 5, 85
Proteintech Group was founded by Dr Jason Li and a group of NIH-funded scientists at the University of Illinois in 2002 when they realised a gap in the market for high-quality, well-validated antibodies in research. The group has over 18 years of experience in this industry and is committed to manufacturing and validating 100% of its products in-house without sourcing from third-party manufacturers. Proteintech’s growing catalog of over 12,000 antibodies is now available in 39 countries across 5 continents, and the group has recently expanded its reach to Southeast Asia.
About the Authors
Mun Wah is a Sales & Marketing executive at Proteintech Group. He graduated with a Bachelor of Science (Hons) in Biological Sciences from Nanyang Technological University. Presently, Mun Wah is working with the Southeast Asia distributors and supporting customers in selecting the best antibodies.
Will Olds is a Scientific Officer at Proteintech Group. He graduated with a Bachelor of Science in Cellular & Molecular Biology from University of Michigan and a PhD in Genetics from Yale University. Presently, Will is providing technical support and advises on the company’s scientific and collaborative strategies. Outside of work, Will is also an avid Venture Fellow in Blue Ivy Ventures, providing consultations for investors in the biotechnology market.