FAQs

  Logo_and_Antibody_image_web.jpg

 

When considering equivalent service items and levels, outside companies cost considerably more than what we charge at the VAPR.  This can be misleading with "special deals" and promotional emails.  Low cost antibody and protein offerings are also typically low service and quality.  To get you the reagent you need takes the level of expertise and attention offered by VAPR.  We never sacrifice quality.

 

​ 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 3-4 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. 

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-$1500) 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. 

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.

  1. Native Protein - 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.
  2. 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.  While generating a high-titer of antibodies to the immunized peptide is relatively easy, it is too often the case that these antibodies show low reactivity to the actual protein of interest.  It has been shown that as much as 50% of the time anti-peptide antibodies show unacceptably low or no recognition of the native protein of interest.
  3. Recombinant Protein Antigens- We find this to be the best balance in terms of obtaining sufficient antigen and in eventual quality of the antibodies produced. Recombinant full-length or protein fragments have a much better track record of generating antibodies that robustly recognize the native protein of interst.  The obvious and most straight-forward approach for many investigators is to generate recombinant antigens in bacteria, but other systems are also avaliable. VAPR can assist researchers in expressing their proteins in bacteria, insect cells, or mammalian cells. The exact system used depends on the protein and the ultimate uses for the antibodies.  Please contact us to learn how we can help produce recombinant proteins for use as immunogens, or any other need.

It is relatively easy to generate anti-peptide antibodies because the technology for peptide synthesis, carrier conjugation, 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. However, in certain cases peptide-based approaches are required.  In these cases, VAPR will perform a full antigen analysis to help you select the optimal peptide to maximize the chances of success.

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.