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Anti-peptide antibody production is an advantage when the antigen (protein) of interest is difficult or expensive to isolate in sufficient quantities, or when a single epitope of the protein has to be detected. Since a peptide molecule in itself is not immunogenic, it must be chemically coupled to a larger molecule (carrier) for successful immunization. Immunized animals will elicit antibody responses, both to the coupled peptide and to the carrier molecule. A common carrier protein of high molecular weight and antigenicity is keyhole limpet haemocyanin (KLH). Carrier-specific antibodies often correspond to a significant fraction of the total antibody pool (10% or more) in the serum of the immunized animal. Presence of anti-KLH antibodies in serum may cause non-specific background, for instance in immunohistochemical staining.
In the plant species, Adonis aestivalis, a specific and distinct staining pattern has been observed that is the result of an immunological cross-reaction between anti-KLH antibodies and an as yet unidentified plant antigen. The cross-reactive antigen is most prominent in the xylem part of vascular tissues. Different parts of the plant as well as other plant species have been tested with similar results.
Therefore, the possibility of cross-reactivity must be seriously considered when planning immunohistochemical localization of plant proteins using antipeptide serum raised with KLH as a carrier. However, the problem may easily be overcome by removing anti-KLH antibodies from the immune serum by passing the serum through an affinity column coupled with KLH. KLH affinity column will efficiently remove anti-KLH antibodies in a single purification step. Thus it is a flexible and convenient way of processing serum irrespective of the peptide specificities. It is also an attractive alternative to conventional purification of specific antibodies on peptide affinity columns, at least for serum where the titres of specific antibodies are high. One may want to consider raising the anti-peptide antibodies using another carrier protein.
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