Antibodies are critical reagents for a variety of molecular and cell biology techniques such as ELISA, western blot and immunohistochemistry. Polyclonal antibodies (pAbs) can offer advantages over monoclonal antibodies when performing assays requiring specificity, signal amplification and broader epitope recognition.
pAbs can be custom rabbit polyclonal antibody against a wide range of targets and are especially useful in proteomic evaluation because they recognize multiple antigens on the same protein. This is important for identifying the small changes in protein structure that can indicate disease-specific biomarkers and help uncover new drug targets.
When developing a custom rabbit polyclonal antibody, the first step is to decide upon an antigen. This can be purified endogenous protein, a recombinant protein or a chemically modified molecule called a hapten. Haptens are small molecules that can stimulate the production of antibodies when coupled to a larger carrier protein such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
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The next step in the process is to prepare the antigen-alum mixture for immunization of the target animal host. The immunization schedule varies with the antigen and animal host species. Typically, the animal will be immunized with Complete Freund’s adjuvant and various aluminum salts referred to as alums in order to optimize the immune response. Several test bleeds are performed over time to monitor the level of specific antibodies produced.
Once the titer of the antisera has reached the desired level, the antibody is prepared for use in an ELISA or western blot. A negative selection method such as Melon Gel Chromatography is used to purify the IgG from crude sera. This simple negative selection technique yields cleaner, higher titer antibody for use in your assay.
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