Polyclonal Antibody Production
A Fast Way to Get the Antibody You Need
Frequently Asked Questions
Are you working with a new protein system for which there is no commercially available antibody? Do you want to purify a new protein or want to investigate protein complex formation by immunoprecipitation of near neighbors? Are you studying phosphorylation of a new type of signal protein? Do you often say, "I wish I had an antibody for that!"?
Then, custom polyclonal antibody production might be for you.
Polyclonal antibodies are used in a wide variety of applications, for example: in ELISA, Western Blot, Immunoprecipitation, Flow Cytometry, Immunofluorescence, and Immunohistochemistry assays.
Polyclonal antibodies are directed against specific antigens, primarily protein antigens. These antibodies are the result of multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Immunogens, or antigens, are any substance, such as protein, lipid, or carbohydrate, which can elicit a humoral immune response. This is in contrast to monoclonal antibodies, which are derived from a single B-cell clone and produce an antibody to a very specific antigen.
To generate polyclonal antibodies, the antigen is intentionally injected into the host. The antigen is processed by the immune response, which ultimately results in proliferation and differentiation of B cells into antibody-secreting cells. Blood serum is then collected and antibodies can be purified further for use in specific assays. To enhance the antibody response and maximize the antibody titer, adjuvants are included with the antigen. Adjuvants are substances that enhance the immune response, generally by creating a depot for slow release of the antigen.
A wide range of antigens can be used as immunogens:
1) Protein based antigens (native or denatured, soluble or insoluble, whole cells or cell lysates)
2) Haptens (peptides or small molecules, such as testosterone or estrogen).
3) Whole Protein Antigens
Generating polyclonal antibodies against a full-length protein will provide a pool of antibodies against multiple epitopes from the protein, thereby maximizing the probability of recognizing the endogenous protein in the target assay. Another advantage of full-length protein immunization is the likelihood of generating an antibody with a broad range of applications (WB, IF, IHC, IP, FC).
The characteristics of a good antigen include:
1) areas of structural stability and unique chemical complexity within the molecule;
2) significant stretches lacking extensive repeating units; and
3) structural elements that are sufficiently different than the host.
The size of protein antigens can dramatically affect the quality and quantity of antibody produced. Small polypeptides (10kD) and non-protein antigens need to be conjugated to larger, immunogenic, carrier proteins to increase immunogenicity. Generally, the larger the immunogenic protein is, the better.
The use of native protein vs. recombinant or denatured protein should be considered when planning antibody production. Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins. If the antibody is to be used in a method that detects denatured protein, as in Western Blot or Immunohistochemistry on fixed tissue, then denatured protein should be used to generate the antibody. If the antibody is to be used in a method that detects native protein, as in Flow Cytometry or Immunoprecipitation, then native protein should be used to generate the antibody.
Antibodies made against recombinant proteins can in some cases fail to recognize native protein. The reason for this might be incorrect folding of the protein antigen when expressed in bacterial cells.
There are a variety of computer programs that help to predict the immungenicity of synthetic peptide antigens (1,2). For peptide sequence selection, it is important to select sequences that are found on the surface of the native protein, contain both hydrophilic and hydrophobic residues, and preferably have antigenic amino acids (e.g. Cys, Leu, Val, Arg, etc.).
To select sequences that are found on the surface of native proteins, the N-terminal and C-terminal regions of a protein are good candidates. C-terminal regions are often species specific, which also helps to narrow the focus of the antibody developed. Other sequences that are known to be included in cytoplasmic loops or extracellular regions are good candidates for peptide sequence.
For peptide length, as a general rule, longer is better. Approximately 8-10 amino acids is generally the smallest peptide used. Peptides of 15-20 residues are the most commonly used. For antigens less than 10kD, which includes all synthetic peptides, the peptide needs to be conjugated to a carrier to generate a significant antibody response. The most commonly used carrier is KLH (Keyhole Limpet Hemocyanin) but albumins, generally BSA and OVA, are also used.
Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site antigen depot, which allows for a slow release of antigen into draining lymph nodes. Many adjuvants also contain or act directly as:
1) surfactants which promote concentration of protein antigens molecules over a large surface area, and
2) immune-stimulatory molecules.
Adjuvants are generally used with soluble protein and peptide antigens to increase antibody titers and induce a prolonged response with accompanying memory.The discovery of adjuvants dates back to 1925 and 1926, when Ramon showed that the antitoxin response to tetanus and diphtheria was increased by injection of these vaccines, together with other compounds such as agar, tapioca, lecithin, starch oil, saponin or even breadcrumbs.
The most frequently used adjuvant used in animals is Freunds Adjuvant. There are two Freunds adjuvants, Freunds Complete Adjuvant (FCA) and Freunds Incomplete Adjuvant (FIA). FCA is composed of heat killed, dried Mycobacterium species added to mineral oil (Bayol F) at 85% and a detergent (Arlacel A) at 10-15%. Freunds complete adjuvant is a potent adjuvant giving good antibody response. FIA contains mineral oil and detergent but does not have Mycobacterium spp included. It is generally used for booster immunizations following initial immunization. FCA causes inflammation, and local granuloma formation and local site lesions may follow injection of FCA or FIA.
1. How much protein is needed?
3-5mg is preferred. For the immunizations for an average project, 2 mg of protein should be enough. This will give enough for an ELISA as well. The final amount needed depends upon how long the project continues. If affinity purification is needed, we would need another 3 mg for the column and testing.
2. Should the protein be supplied in native or denatured form?
The protein does not need to be denatured. It is important to avoid detergents in the protein sample as they will decrease the stability of the adjuvant emulsion and could result in lower immune response.
3. Can the protein be provided as SDS-PAGE gel slices?
Gel bands are OK to use. They can be stained and destained, and washed well with water to remove the acetic acid and methanol. It is best to provide the whole strip, in a 15ml conical tube, rather than breaking it up. We would need to know the approximate protein content; it would be best if there were at least 100ug of protein in 200ul of gel.
4. What buffers and concentrations are best for the immunogen?
The protein should be in a mild buffer such as PBS. The best concentration range is 0.5-1.0mg/ml. If it is more concentrated we can dilute accordingly.
5. My antigen is not soluble. What should I do?
Usually we can inject an insoluble antigen. If the particles can fit through an eighteen-gauge needle, it will work. We recommend not using detergents such as SDS to make the antigen soluble. We will vortex the antigen/buffer mixture and make an emulsion with the adjuvant to ensure a homogeneous preparation
6. What happens if my antigen preparation is not pure?
The animal will make antibodies to whatever is injected. If the antibodies to contaminating proteins or other components may cause a problem, consider an alternative method. We recommend that the purest material be injected first.
7. How many animals should I use?
We recommend that at least two, preferably three animals of each species for antibody production. We have found variability of the antigenic response in different individuals. Using more than one animal allows a more diverse response in terms of quantity of antibodies, specificity, and affinity. However, one animal is appropriate if the antigen has been used in the past successfully to raise antibodies.
8. I think I need to conjugate my peptide. What should I use?
There are many conjugates. We recommend that small peptides (less than 15kD) and nonprotein antigens need to be conjugated to a large immunogenic carrier protein. The most frequently used is keyhole limpet hemocyanin (KLH). Others are bovine serum albumin (BSA), thyroglobin, ovalbumin, and rabbit serum albumin (RSA). Conjugation is not a substitute for using an adjuvant. The adjuvant helps concentrate and deliver the antigen to the immune system. Conjugation increases immunogenic properties of the antigen by increasing the size of the molecule.
1. Kolaskar, A.S., Tongaonkar, P.C., "A semi-empirical method for prediction of antigenic determinants on protein antigens."FEBS Lett. 6:172-174 (1990)
2. Hopp, T.P. "Retrospective: 12 years of antigenic determinant predictions, and more." Pept. Res. 6:183-190, (1993).