Antibodies, also known as immunoglobulins, are relatively large macromolecular glycoprotein structures produced by a host organism that recognize, bind, and facilitate an immune response against foreign material (antigen). The antibody is the basic functional unit of the adaptive immunological response.
Canonically represented as a split Y-shaped structure, antibodies are composed from of two identical “heavy chains” that make up the Y-resembling structure and two smaller “light chains” that flank the arms of the Y. Heavy and light chains are held together by a series of disulfide bonds, which permit the antibody to retain it‘s commonly identifiable superstructure. On opposing ends of the structure, heavy and light chains combine to generate a region known as the F(ab) or Fragment of Antigen Binding, a highly a variable region that is responsible for recognizing and binding foreign material (see figure 1).
The F(ab) region
The exceptional variability of the F(ab) region contributes to the diversity of the immune system, allowing production of an array of different antibodies that are highly specific individually, but are collectively capable of recognizing billions of different antigenic structures. In a serum sample, any given individual antibody will be highly specific for a relatively small segment of its respective antigen(known as an epitope). However, the bulk of antibodies in circulation against an antigen will recognize a broad range of different epitopes.
Different classes of immunoglobulins are often responsible for facilitating immune response to different types of antigenic targets, and are present in different tissues and fluids. Some immunoglobulins exist solely as monomers, while others can be found as multimeric superstructures composed of several individual monomers joined together.
Antibodies for research applications
Antibodies have always played a pivotal role in the study of molecular and cell biology. It was no significant stretch for life-science and biomedical researchers, already intricately familiar with immunology, to co-opt the flexibility of the adaptive immune response to bind, precipitate, collect, or label their protein of interest with a high degree of specificity. Today, the affinity proteomics market (encompassing commercially available antibodies for research and other products derived from them) is a booming industry, generating billions of dollars of revenue worldwide. While development of proteomics reagents has advanced, the basic technique for production of most antibodies today is strikingly similar to the process used when they were first introduced into the research realm. Antibodies sold commercially for use in research today fall into one of three general categories:
The most common and inexpensive antibodies on the market are polyclonal antibodies. Polyclonal antibodies are a heterogeneous mix of antibodies, collected from serum, generated against a specific target. Commercially available polyclonal antibodies come from many host sources (e.g. rabbits, goats, mice or other animals) but the general protocol used to generate them is similar regardless of host:
- Couple a target of interest to a compound that will prompt a large immune response in a host animal (known as an adjuvant or carrier-protein).
- Inject the conjugated mixture into a host animal to drive an immune response.
- Collect serum from the host animal.
- (Optional) purify antibodies from the serum.
Polyclonal antibodies carry the benefit of being simple, inexpensive, and quick to generate. However, they generally lack the specificity of a monoclonal antibody. For long-running experiments there are also supply chain concerns with polylclonal antibodies. As each heterogeneous mixture of polyclonal antibodies generated will be slightly different, attempts to recreate a specific lot of polyclonal from a different animal (or even the same) may produce unpredictable results.
Monoclonal antibodies are the second most common form of antibody found in the research market. Monoclonal antibodies are more difficult and labor intensive to create, but are more specifically targeted and do not commonly suffer from the supply chain issue that polyclonal antibodies do. Monoclonal antibodies are produced by:
- Immunizing a host animal, as with a polyclonal antibody.
- Collecting antibody producing spleen cells from the host animal.
- Fusing these spleen cells with myeloma cells to generate an immortalized cell line (a hybridoma).
- Collecting and purifying antibodies produced by this cell line.
Monoclonal antibodies are, by definition, specific for a single epitope. All antibodies produced from the same hybridoma clone will recognize the same region of a target. Additionally, because the cell line is immortalized a given hybridoma cell line can continue to produce antibodies indefinitely, negating concerns related to future antibody availability. However, the increased specificity and peace of mind does come with a cost, commonly in the form of a price tag. Monoclonal antibodies are often more expensive than their polyclonal counterparts, due to the labor-intensive process necessary for their creation. Their high degree of specificity can also have a net negative impact for a certain applications.
Of the three classes, recombinant antibodies are the most recent and the least prevalent in the marketplace. Recombinant antibody production leverages advanced molecular biology techniques, allowing researchers to clone genetic sequences for antibody production into expression vectors and co-opt simple organisms like viruses, yeast or bacteria to produce antibodies for them. Today, recombinant antibodies are still fairly expensive, and are only available against a small selection of targets. This is due in part to their novel nature and the R&D overhead that goes into creating them. Looking to the future however, recombinant technologies do offer an exceptional opportunity for large scale, rapid, inexpensive, and simple antibody production.
Recombinant antibody production has given rise to a number of valuable new technologies, allowing experimental approaches that were not previously possible. For example, “nanobodies” produced by ChromoTek GmbH are recombinant alpaca antibodies that express only the smallest functional unit of the antibody, the antigen binding fragment. Researchers can employ these tiny “nanobodies” in experiments where use of conventional antibodies would not be a feasible option.
Navigating the antibody marketplace
Prevalent in life-science research for more than 70 years, and utilized in hundreds of different applications, antibodies of all varieties are among the most widely used, versatile, and accepted research reagents. Commercial antibodies are more available today than ever before, but the booming marketplace has brought a number of significant difficulties. Researchers planning or conducting an experiment are often fraught with challenges presented by a market that lacks transparency and accountability.
Antibodies are commonly sold to end users, not by original manufacturers, but by distributors. Many of these distributors also sell to each other, and a significant quantity relabel the products that they sell in an attempt to promote their own brand. The result is that the original developer of a specific product is not commonly discernable by the time it reaches the end user.
The industry is also plagued with issues pertaining to the quality and validity of antibodies that are produced and brought to market. The rise in the number of viable antigenic targets discovered through rapidly advancing genomics research, coupled with ever-increasing pressure on academic researchers to “publish-or-perish” has driven production of antibodies against novel targets to an all-time high. Regrettably, in this fervor, new products are often not subjected to the rigorous quality control standards that are commonly expected, and researchers often report having to trudge through a deluge of low-quality ineffective proteomics reagents before finding one that will do the job.
In response to reports of these severe difficulties a select number of researchers, reagent manufacturers, and distributors have stepped up with innovative solutions. In 2013, MIT based Science Exchange partnered with antibodies-online, a key distributor of antibodies and related products, to kickstart the Independent Validation Initiative (http://www.antibodies-online.com/independent_validation/). Through this initiative, high-demand products are supplied to independent laboratories in the United States, and are thoroughly tested for efficacy in the applications listed on their datasheet. Upon approval, the product is awarded a unique identification number and is approved as “independently validated”. Complete validation reports for all validated products, including detailed protocols and results, are published on the antibodies-online website for potential purchasers to review.
Tips for buying and applying antibodies
Part of what makes dissecting the problematic reagent market difficult, is that the application of antibodies in any experiment is a complex and nuanced procedure. It is often difficult for an independent observer to clearly and accurately distinguish whether a reagent is at fault, or whether user error and a poor protocol are to blame. Here are a few helpful dos and do nots keep in mind when purchasing and applying antibodies to help minimize frustration during an experiment:
- Do plan ahead: Have a clear idea of the sort of experiment you want to perform. What will you need? Do you need a highly specific monoclonal antibody, or will a polyclonal do? Will you use a directly labeled primary antibody, or employ a secondary antibody? How will your sample be prepared? Many antibodies are only validated for use on certain types of sample preparations.
- Do your homework ahead of time: information is currency in the antibody world. Know which applications an antibody is approved and guaranteed for. Know the species reactivity that are approved, and know which other reagents are compatible with the antibody in question.
- Don’t be afraid to ask questions: Most reagent manufacturers and distributors staff full-time, doctoral level support professionals who are there to guide customers and dig up information on a product that might not be readily available. As with all avenues of science, it is always better to ask than to guess.
- Do remember that antibodies are proteins: Avoid conditions that would result in protein denaturation or destruction. Extreme temperatures, highly acidic or basic conditions, or the presence of strong detergents can and will inhibit antibody function.
- Do use reliable controls: If no experiment is truly a failure unless you learn nothing from it, then good controls are the defining factor between a failed and a successful experiment. Controls will be context and experimentally dependent. (i.e. you’ll use a different set of positive and negative controls for a western blot than you will for an IHC experiment). Refer to tip #3 if you don’t know the right controls to use.
- Don’t assume that more is always better: Antibodies work best at specific concentrations. Remaining within the manufacturer suggested guidelines for dilution, or performing a set of experiments to determine the optimal concentration is more likely to yield success than just “adding more antibody”.
- Do consider using a previously validated reagent: It’s not always an option, but if you can find an antibody that has been used against your target in a publication, or better yet one that has undergone stringent independent validation you can save a tremendous amount of frustration and difficulty. Time that you aren’t spending optimizing and sorting through poor quality reagents is time that you could be spending preparing, performing, or publishing an experiment.
The future of research antibodies
Use of antibodies as tools for basic science and biomedical research is longstanding tradition. Despite continual advancement in research technology the use of antibodies is not predicted to dissipate anytime soon. Instead, modern methods and protocols lean toward optimization and improvement traditional affinity proteomics techniques. Today new tools and techniques for development and application of research antibodies have coalesced with a modern web-based marketplace where researchers can rapidly view, compare, and purchase millions of different offerings from hundreds of manufacturers in moments without leaving the laboratory.
While modernization of the research antibody market has been stymied by a number of “growing pains” few can argue the fact that advancement in technology with regards to locating, purchasing, and applying reagents has been a boon to the research community. As with any laboratory purchase, if a researcher proceeds with caution, and tempers optimism regarding new reagents with a healthy dose of rigorous, logical skepticism he or she can navigate the market and find a variety of products that are suitable to fit nearly any experimental need.