Companies cost between 2 and 3 times more than what we charge at the VAPR. More importantly, we’re right here at Vanderbilt - we can work with you to discuss, design and execute relatively sophisticated strategies that would not be feasible for a company, and we can troubleshoot together if unexpected problems arise. Because of the distance and complications involved in mailing samples, companies generally offer little more than a guarantee to provide ELISA positive clones to a purified antigen. You send them purified antigen, and 5 months later (if things work out), they send back 5-10 clones that score highly by ELISA. The antibodies may or may not do what you need. We can work directly with you to vastly increase the odds of getting the right antibodies. If you need mAbs that work in a particular assay (eg, immunoprecipitation, immunofluorescence, Western blotting ,Immunohistochemistry), we can work with you to screen specifically for these functions.
Although nobody can provide an absolute guarantee in this business,there is quite a lot that we can do to optimize your chances of success. We will start with a meeting where we discuss your goals and design an experimental approach that is selectively tailored to your particular project. For example, if you have had problems in the past with generating a strong immune response, we can immunize several different mouse strains (or rats) to enhance the odds of finding a host that responds appropriately to your antigen. Moreover, a typical fusion will yield between 10 and 100 positive clones, and we can coordinate efforts to make it possible for you to screen all of these clones in order to isolate rare clones with specific properties. Frequently, such clones are not the same as those that score the best by ELISA.
Finally, we have set up the Vanderbilt Antibody Repository (or VAR) to provide a mechanism for distributing Vanderbilt antibodies to others. If you are interested in distributing your antibody through the VAR, we can return some of the revenue to you as a royalty. It is likely that the cost to your lab of generating the antibodies can be entirely recovered in a relatively short period of time. We will handle quality control and maintain a website that describes the properties of each antibody in detail. Because our overhead is low, we can distribute the antibodies at about half the cost of a conventional business. The VAR will run like a nonprofit business with cost structures designed to offset and/or subsidize the VAPR. Vanderbilt investigators are encouraged to use this mechanism as a way to generate collections of high quality antibodies of interest to particular consortiums or groups.
If you already have a purified antigen, it generally takes about 5 months, depending primarily on how many times you have to boost mice to get a good immune response. The various steps and associated considerations are outlined in the flow diagram under Technical Resources. In a typical project, immunization takes 2 months. Another month is required for fusion and screening. Positive clones are usually subcloned twice, which requires another 4 - 6 weeks. Sometime animals have to be immunized a 3rd time if the animals respond poorly to the target antigen, which adds a month to the overall time. For many labs, the main obstacle is in fact the preparation of the antigen itself. Click here for information on the initial generation and purification of antigens and related issues.
Because of the time required to make monoclonals, it is worth weighing the pro’s and con’s of generating monoclonal versus polyclonal antibodies. In general, the specificity and unlimited availability of monoclonal antibodies is worth the additional time and effort, but rabbit polyclonal antibodies are a reasonable and sometimes preferable alternative, depending on your specific needs.
Polyclonal antibodies (2 rabbits for ~$1000) can be generated in about 2 months by sending purified antigen to various companies that specialize in this technology. They will immunize the animals and send you the bleeds as they become available. If you are able to generate 4-5 mg of extremely pure antigen, this may be the best option. It’s fast, relatively inexpensive, and can yield large quantities of high titer polyclonal antisera. Indeed, a good high titer polyclonal antisera can be superior to mAbs in terms of signal intensity because the antibodies recognize multiple epitopes. This is a particularly good option if you need antibodies fast and are not overly concerned with specificity. For example, if you need only to be able to visualize the protein on a Western blot and you know its size, then a polyclonal antisera is ideal. However, the same antisera might be useless for immunofluorescence if it interacts with multiple bands on a Western blot. Click here to see a list of recommended custom polyclonal antibody companies.
On the downside, each time the animal is boosted, the character and titer of the antisera will change. Therefore, in principle, and often in practice, each bleed needs to be characterized separately. If the immunogen is not extremely pure, antibodies to contaminants will also be present. Contaminants can be binding partners that copurify with the antigen making it impossible to use the antisera for validating protein-protein interactions. Even if the antigen is pure, the recognition of multiple epitopes by polyclonal antibodies increases the chances of unwanted cross reactivity with other proteins. This kind of problem is rarely encountered with monoclonal antibodies.
The main advantage of a monoclonal antibody is specificity. Although more difficult to generate, a monoclonal represents an unlimited source of highly specific antibody that can be easily purified. For most, the specificity and ease of use of mAbs outweighs the additional cost and effort. For anyone involved in significant long-term study of a protein, monoclonal antibodies are generally the way to go.
The technology itself has advantages that may not be immediately obvious. For example, the antigen preparation does not have to be pure - you can inject a partially purified protein mixture, or a protein complex that contains your antigen, and then screen later for clones that specifically recognize your antigen or various other components of a protein complex.
Many believe that the best antigens are native proteins because they are correctly folded and provide optimal templates for antibodies that might recognize different kinds of determinants such as conformational (good for immunoprecipitation) and linear (good for Western blotting) epitopes. In practice, it is difficult or impossible for most labs to purify adequate amounts of native protein from biological mixtures. Therefore, antigens are generally prepared by recombinant technologies (ie, expression of 6XHIS-tagged proteins in bacteria) or by chemically synthesizing peptide antigens.
Peptide antigens have a couple of obvious advantages. They are easy to generate and by definition define the antibody binding site on the target protein. If you want to target a particular defined epitope within a protein, a peptide antigen is the obvious way to go. Peptides work best if they are greater than 12 amino acids in length, and must be conjugated to an appropriate carrier protein (eg, BSA or KLH) in order to generate a significant immune response. Shorter peptides are adequate if one uses a chemical spacer arm to distance the peptide from the protein carrier. An important modification of this technology is the use of phospho-peptides to generate antibodies that selectively interact with the phospho-epitope. These so called phospho-specific monoclonal antibodies are powerful tools for analysis of signaling events regulated by phosphorylation.
There are a couple of potential disadvantages of peptide antibodies. Frequently, the affinity of antisera to peptide antibodies is low. It might be possible to get around this by generating monoclonals, in which case one can sometime isolate a high affinity monoclonal antibody. Also, it is sometimes difficult to know whether a particular peptide is exposed on the surface of a protein in such a way as to be accessible to antibodies. If not, the antibodies will only work in assays such as Western blotting of SDS-denatured proteins. There are practical guidelines for designing peptides for antigens, which include methods for predicting solubility and the likelihood that the fragment will be exposed on the protein surface.
The obvious and most straight-forward approach for many investigators is to generate recombinant antigens in bacteria. There are quite a few powerful vector systems that facilitate the cloning of your favorite cDNA fragment in frame with various tags that are specifically designed to facilitate purification (eg, 6XHIS, GST, etc.). Many of these are combined with unique protease digestion sites that allow one to cleave off the tag following purification. The Vanderbilt Structural Biology Core provides an outstanding collection of such vectors (click here), and expert assistance in all aspects of their use.
It is relatively easy to generate anti-peptide antibodies because the technology for peptide synthesis, carrier conjugtion, and immunization is well established. On the other hand, these antibodies are often of relatively low affinity and can be ineffective in recognizing native proteins if the peptide is not exposed on the protein surface. Please see the peptide design and synthesis information within the Monoclonal Production Process Flowchart for information relevant to choosing favorable peptide sequences and peptide synthesis.
Phosphospecific monoclonal antibodies can be somewhat more difficult to generate than conventional anti-peptide antibodies and require additional screening. On the other hand, the technology is not different from conventional methods of generating monoclonals, and some phosphoepitopes are quite immunogenic. It is sometimes difficult to get phosphospecific antibodies that do not show at least limited cross reactivity with phosphoepitopes on other proteins. This is presumably because a key element of the epitope (ie, the S, T, or Y phosphate group) is common to many other phosph-proteins. Although these issues present special challenges, the overall success rate is reasonable, and these antibodies can be extraordinarily valuable in signaling studies.
Mouse monoclonal antibodies come in several classes (eg, IgM, IgG, etc). Because IgM antibodies are of low affinity and hard to work with, the immunization protocols are designed to avoid IgM antibodies and give mostly an IgG response. There are several different “isotypes” of IgG that are structurally (and functionally) similar but differ from one another by the presence or absence of a few antigenic determinants. All of the IgG isotypes work great for typical biologic assays such as IP, WB, and IF. About 90% of the monoclonals derived from a typical fusion will be IgG1, with the remainder being IgG2a, IgG2b, or IgG3. Though mostly irrelevant in terms of how they function in an assay, it can be important to know the isotype in particular situations. For example, the different isotypes bind to protein A and protein G with markedly affinities at markedly different pH’s, and are therefore relevant to elution strategies used for the most common antibody purification methods (which are based on affinity purification of IgG antibodies on Proteins A or G affinity columns). More importantly, companies offer quite a few “isotype” specific secondary antibodies conjugated to fluorescent dyes. These can in turn can be used in double immunofluorescence experiments to distinguish between proteins recognized by primary antibodies of different isotype.
Optimal storage depends on what you plan to do with them. Antibodies are reasonably stable and can be stored for weeks at 4˚C without noticeable loss of activity. However, for storage longer than a couple of weeks, one should either freeze them, or keep them in 50% glycerol at -20˚C. The main consideration is that antibody preparations in general lose about 10% of their activity when they are frozen and thawed. Therefore, any strategy for storage and use should aim to limit freezing and thawing. For example, serum should be aliquoted before freezing so as to avoid repetitive freezing and thawing. For routine lab use in procedures such as immunoprecipitation, Western blotting, and immunofluorescence, it is best to dilute the antibody preparations in 50% glycerol for storage at -20˚C. The preparation is perfectly stable for many years in this condition, and because it does not freeze, one can repeatedly pipette directly from the vial without ever having to subject the sample to freeze/thaw cycles. For applications where dilution in glycerol is unacceptable, antibodies should be aliquoted in single use amounts and frozen. -20˚C is OK, but -80˚C is better, particularly for long-term storage.