> Information Center > Technical FAQs > Antibody Technology Column > Where can I find commercial antibodies
Despite an impressive growth in the business of research antibodies a general lack of trust in commercial antibodies remains in place. A variety of issues, each one potentially causing an antibody to fail, underpin the frustrations that scientists endure. Lots of money goes to waste in buying and trying one failing antibody after the other without realizing all the pitfalls that come with the product: Antibodies can get inactivated, both the biological material and the assay itself can potentially be flawed, a single antibody featuring in many different catalogues can be deemed as a set of different products, and a bad choice of antibody type, wrong dilutions, and lack of proper validation can all jeopardize the intended experiments. Antibodies endorsed by scientific research papers do not always meet the scientist’s requirements either due to flawed specifications, or due to batch-to-batch variations. Antibodies can be found with Quality Control data obtained from previous batches that no longer represent the batch on sale. In addition, one cannot assume that every antibody is fit for every application. The best chance of success is to try an antibody that already was confirmed to perform correctly in the required platform.
Based on feedback from about 10 years ago, scepticism and mistrust towards commercial antibodies was already commonplace. Researchers in the academic environment preferred generating antibodies in-house by making use of the animal facilities in their faculties. At the time, the availability of commercial antibodies was not as extensive as it is today, and therefore it was unlikely that a scientist would find an antibody fitting their requirements. The present situation is quite different, yet the complaints remain. The number of commercial antibodies has escalated in the last decade, and so has demand. In contrast to 10 years ago when Western Blot (WB), ELISA and ImmunoHistoChemistry (IHC) were the most used assay types, at present antibodies are increasingly used in more sophisticated platforms such as flow cytometry, multiplex assays, immune-mass spectrometry and other capture-based assays as modern technologies have made them widely accessible. Along with this increased variety of platforms, demand for fit-for-purpose (F4P) antibodies is increasing, while disappointment by the performance of commercial antibodies remains an ever present experience.
Despite the negativity described above, the complexity of generating F4P antibodies has made the research-antibody trade one of the fastest growing markets in the life science industry. Not only has the number of traders increased, the traders also enjoyed a substantial growth in their business. There seems to be no stop in the increasing demand for commercial antibodies for research purposes. Yet, even today, the complaints of poor performance remain the biggest problem in the research antibody industry. Attempts to release multiple antibodies targeting the same protein did not make much of a difference so far. The reasons for this are outlined below.
The scientific community is struggling with the complexity that research antibodies bring to the lab, and therefore each complicating factor is discussed separately before we can build a general picture of how to benefit optimally from commercial antibodies.
The term non-specificity is used when an antibody binds to unintended proteins. Each antibody molecule has a certain affinity to one part of the protein called an epitope, and this affinity is determined by the epitope’s amino acid sequence. It is therefore very difficult to find antibodies that react exclusively to one protein when this protein is very similar to other (closely related) proteins. Only antibodies that will bind to a unique epitope will react specifically to its intended target protein. However, most antibodies do not bind to unique epitopes and so they will cross-react.
In the case of shared epitopes between closely related proteins cross-reactivity is inevitable. Then the actual binding of the antibody may be specific, yet the antibody is deemed non-specific in relation to the intended target protein. Further diluting the antibody and optimizing blocking conditions will not work in these cases. In other words, the specificity of an antibody relies on the uniqueness of the protein part it binds to (i.e. the epitope).
An antibody specific to an epitope that is shared between one or two other (closely related) proteins may not be useless. It may still be useful in tissues or cell types where those cross-reacting proteins are not present. Or the scientist can take advantage in relating the intensities of bands representing the different proteins in Western blot.
Proteins unrelated to the intended target protein may have epitopes similar but not identical to the specific epitopes. Then the antibody’s affinity for the similar epitope will be lower than for the specific epitope. This will result in cross-reactivity with a proportionate lower signal, called non-specific background.
Non-specific background can be reduced by further diluting the antibody. The reason is simple: by diluting the antibody only higher affinity interactions are sustained. The lower affinity interactions (to remotely similar epitopes) will not last at lower antibody concentrations. Proper blocking conditions can also help to prevent low affinity interactions. NaCl will interfere with weak hydrostatic interactions while non-ionic detergent (for example Tween-20) will interfere with weak hydrophobic interactions between the antibodies and the unintended target proteins. Increasing the concentrations of such blocking agents may help to reduce the non-specific background.
Poor experimental conditions will incur random noise and this is typically not related to the primary antibody. Lacking certain blocking components or the use of dirty containers/contaminated buffers are usually to blame. Especially in fluorescence-based assays, noise can be a big issue. There is a risk of antibodies being dismissed prematurely because non-specific background and noise are not considered and dealt with separately.
In addition to primary antibody-derived issues, the secondary antibody can be a source of problems as well. The quality of the secondary antibody can be tested by side-by-side comparison of a complete experiment with another experiment lacking the primary antibody. Noise and background caused by the secondary antibody will become apparent in this negative control.
The term “polyclonal antibodies” (pabs, as opposed to mabs for monoclonal antibodies) is ambiguous and can be the cause of some confusion. There are pabs raised against the full length protein, pabs raised against large protein fragments and pabs raised against small peptides. In addition, there are differences between antiserum, proteinA/G-purified, and antigen affinity purified pabs.
The general concept of pabs is that they represent a collection of antibodies raised against multiple epitopes and possibly against multiple proteins. When an antibody is raised to an entire protein, it is easy to see how multiple parts of the protein will generate a collection of different specificities and affinities. This is beneficial for certain applications such as immune precipitation (IP) and WB where cross-reactivity is easily spotted by the difference in molecular weight (unless there is cross-reactivity to proteins with the same molecular weight) compared to the intended target protein. Pabs can also be useful in IHC, as long as there is no cross-reaction with any other proteins present in the tissue sections of interest. The specificity of an antibody can be addressed by comparing endogenous expression levels to knock-down expression levels, comparing un-induced cells to induced cells (with elevated expression levels) or by looking at tissues where the location of the protein of interest is expected in one particular compartment or cell type.
Antibodies raised against a protein fragment will show higher specificity when the amino acid sequence of the fragment is unique in the proteome. Then the above mentioned advantages of pabs are combined with the uniqueness of the antigen. Although such antibodies may not compete with the mono-specific characteristics of mabs, they can work together with mabs in sandwich type ELISA and IP; using one as the capturer and the other as the reporter.
When the chosen antigen is a small peptide of the size of an epitope instead of a protein or a protein fragment, then the mono-epitopic characteristics of mabs are approached. It would be a prerequisite for this approach to have the peptide antibodies affinity purified using the antigen, thus giving high affinity pabs the upper hand over low affinity mabs. The cost of generating peptide-specific antibodies is also competitive with the cost of generating mabs because neither expensive screening and sub-cloning, nor antigen purification steps are required. A 10–15 amino acid peptide is easily and quickly synthesised and delivered by a specialist service. Just linking the peptide to a carrier protein and dialysis is required to have the material ready for immunization. And the peptide will subsequently be used for the affinity purification.
Peptide antibodies therefore are versatile tools that combine the mono-specificity attributed to the mabs with the high affinity attributed to polyclonal antibody while keeping the production costs low. In addition, the epitope (peptide sequence) for a peptide antibody is known from the product sheet, while caution is required when a mab is used without its epitope characterized and/or published. The one advantage of mabs over peptide antibodies is the mab’s longevity: As long as the hybridoma clone remains stable, the exact same antibody will be generated. This makes monoclonal antibodies preferred over pabs for commercial kits.
Antiserum and protein A/protein G purified IgG still have a mixture of affinities and specificities. From all pabs, only antigen-affinity purified antibodies will have the highest grade of specificity and affinity, and particularly so when they are peptide-derived. In my opinion peptide antibodies are ideal for research purposes, while monoclonal antibodies are ideal for long-term repeated standard assays. Yet, peptide antibodies serve as a (temporary) alternative as long as a proper mab is not available for the standard assays.
The vast majority of vendors do not manufacture all the antibodies on the catalogue, and most of their antibodies have been obtained from a wide variety of different manufacturers from all over the world under OEM agreement (Other External Manufacturer). Such an agreement usually has a clause to forbid the supplier from publishing which of their products are sold by their OEM vendor. The vendors keep up the appearance that they themselves are the primary source of all their antibodies. This enables them to keep QC data on the product sheet that were generated many years ago thus keeping the sales going, while the actual antibody that generated these data may have sold out and has been replaced by successive other batches (from different animals) and the current batch on sale may no longer be able to generate such data at all.
Even monoclonal antibodies suffer from batch-to-batch variations, but not to such severe extent as some types (see above) of polyclonal antibodies. Nonetheless, certain hybridoma clone numbers are still being used for decades while, just like with cell lines, hybridomas cannot be the same after so many passages anymore. It is therefore misleading to use QC data that were generated decades ago, unless the current batch has proven to still be capable of generating such data (in which case one might as well show the latest version of the data).
Assay developers are advised to buy antibodies straight from the manufacturer and ask for a free validation sample from a large batch in stock. Once validated for the required platform, the same batch then can be purchased in bulk so to prevent batch-to-batch variations during the entire project. Identifying the manufacturer can be a challenge though, and the only way to find them is to start looking for overlap between the product sheets from the different vendors. This way, a list can be generated at which point one can guess who the manufacturer was based on additional details still present on the manufacturer’s product sheet but not elsewhere.
Vendors accrue data from their own customers or from their own QC department, thus making the OEM product look unique. This way the same antibody can show different QC data on different catalogues. And while batches run out and are being replaced by others, it can happen that a vendor has still some of the old batch in stock, while another vendor will keep the QC data obtained from the former batch on their product sheet. From this moment on customers start to buy products that are no longer necessarily reflected by their product sheet.
Vendors do not only obtain their antibodies from the original manufacturers. There is a network of vendors obtaining each other’s catalogue items. Consequently, the same antibody starts to occur several times in one catalogue: one time with the current QC data provided by the original manufacturer, and one or more times with QC data obtained from the other vendor’s direct customers or QC department. Potentially, assay developers buy several antibodies from several vendors thinking they are buying different antibodies, yet a number of them originate from the same manufacturer’s catalogue number.
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