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Polyclonal Antibody : Antigen Design

epitope prediction) is not merely to find a peptide that can generate strong immuno-response protein. Antigen design is a very difficult area that has been subject to extensive study, but still far from being fully understood.

In general, a good peptide antigen has the following properties: protein surface location, flexible (usually loop) rather than helical structure, complicate and unique sequence, not a post-translational modification site or functional site (unless the antibody is intended to recognize such a site) and easy for synthesis. But the quantitative relationship between these properties and antigenic strength is not clear.

Antigen design is usually done by two different approaches: predicting peptide's physical- chemistry properties or making prediction based on statistic results. Both approaches have their limitations. Physical chemistry properties such as peptide location in a protein or its secondary structure, particular turn, are difficult to be predicted with high degree of accuracy This is because the problem themselves are parts of one of the most challenge areas of modern science ---- protein folding. Another problem is that in most cases, an isolated peptide in solution can not maintain its native conformation found in the protein. This is the major reason that antigens designed based on known 3D structures are also often failed. Statistic methods that base on frequencies of amino acids in known epitopes fare even worse. The predicted results are marginally better than randomly picked sequences, according to a recent study (Blythe M. and Flower D. (2005) Protein Sci., 14:246-248).

The method by Kolaskar and Tongaonkar combines both approaches and is one of the most popular epitope prediction methods. They claim that its accuracy is up to 75% (Kolaskar AS & Tongaonkar PC (1990), FEBS Lett. 276: 172-4)

Antigen design can be further complicated by the application of an antibody. The above methods are for antibodies intended to recognize the protein in its native folded conformation, which is suitable for experiments such as immuno-precipitation, cell image and other structure and function studies as well as vaccines. It may not work for SDS PAGE based Western blot. The conformation of a SDS-denatured protein is different from its native one. It is thought to become cigarette-like shape with hydrophobic residues being exposed to the outside. SDS molecules bind to the hydrophobic surface. Exactly how the structure looks like is not clear so far. Attempting to obtain such structure has not been successful due to technical difficulties.

The antigen design service provided by EZBiolab is consist of three parts: 1. Use several popular publicly available programs 2. Narrow and refine the results from part 1 with our own algorithm ( the principle is based on Kolaskar and Tongaonkar but with incorporation of some parameters that are derived from our own experiences) 3. Blast search to filter out similar sequences

EZBiolab is more than glad to provide antigen design assistance to its customers. At the same time, we strongly encourage our customers to design their own antigens because knowledge about the structure, function, bioinformatics and other properties of each protein is essential for finding a good antigen. Customers know their own proteins much better than we do. If a customer is not familiar with this subject, we can provide several potential peptides based on the general principles to allow him/her make the final decision.

Again, we like to emphasize that antigen design is not to find a peptide that can generate strong immuno-reaction from animals. It is to find a peptide that generates an antibody to specifically react with its target protein. In fact, over emphasis ELISA titer to the peptide can generate the opposite results. An example is trans-membrane domain peptides. These peptides usually cause strong immuno-responses but they are obviously poor antigens from the standpoint of protein-binding. For this reason, EZBiolab does not provide full guarantee to our antibody service by promising high ELISA titers to peptide antigens. We believe such guarantees do not mean much and to a large extent, is misleading. By no means a strong anti-peptide antibody can be assured to also bind to its protein strongly and specifically.

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