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Chemical synthesis of peptide antigen is a small molecule, itself is difficult to have good antigenicity, only very weak immune responses induced the animals, so it is very important and carrier protein crosslinking. Carrier protein contains a lot of epitope, stimulating T - help cells, and induced B - cell reaction. For there are many, and the carrier protein peptide coupling is one of the most common keyhole limpet hemacyanin (KLH), bovine serum albumin (bovine serum albumin, BSA) and ovalbumin, ovalbumin (OVA) and cattle thyroglobulin (bovine thyroglobulin, THY). KLH has higher antigenicity, is the most commonly used peptide coupling carrier. As peptide carrier BSA is common, but because of the BSA is often used as test blocker that produced by the method of the antibody there is certain limitation in application.
To generate a good antibody, must choose and antigen source different animals. In order to get the biggest immune response, absolutely can't let the immune animals' self recognition on antigen. For example, to generate antibodies against human protein, using the rabbit or the mouse is better than using monkeys. For a very conservative mammalian proteins, it is better to use birds animal (e.g., chicken).
We usually use freund's adjuvant (Freunds) mixed with antigen. First injection using Freunds completely immune adjuvant. Used in combination with adjuvant and antigen, can improve the immune response, and with less vaccine can generate strong immune response. Adjuvants can make slow release of antigen, so as to produce constant stimulation. Hypodermic injection way for multiple sites. Each injection animals blood samples collected immune, to and immune blood sample after comparison. Collected serum samples containing different immune type and subtype.
Detection of polypeptide responses are the easiest way is to anti peptide ELISA. There are two kinds of technology of peptides can be linked to ELISA plate. The first method is to peptide link directly to tray. But if the link just amino acid is part of the antigenic determinant, antibodies cannot and polypeptide further links, so the possibility of false negative results. The second method is the first peptide coupled with carrier protein, and polypeptide protein - I fit link to ELISA plate. This link method can make antibodies - the combination of the peptides to improve success rate, because the peptide is not directly embedded plate membrane, thus will be more exposed to the antibodies. This method is more reliable, higher repeatability. It is important to note that for carrier protein must be different from the method for immune coupling carrier protein. This will avoid serum anti carrier protein reaction caused by the high background.