Polyclonal antibodies were raised against membrane-associated calcium-binding proteins (apparent molecular masses 65000 and 67000 (CBP 65/67) and 33000 and 35000 (CBP 33 and CBP 35)), which were isolated from rat liver and Morris hepatoma. Using immunoblotting, various amounts of CBP 33 and CBP 35 as well as CBP 65/67 were detected in most rat organs. Using alkaline phosphatase and monoclonal-anti-alkaline phosphatase antibodies (APAAP), all the calcium-binding proteins were detected by immunohistochemical techniques in the plasma membranes of many cells, such as vascular endothelial cells, lymphocytes, epididymal principal cells, secretory and excretory duct cells of certain exocrine glands, straight distal tubular cells of the kidney, and in the cytoplasm of muscle cells and fibres as well as nerve cells and chondrocytes, and in connective tissue elements. Immunohistochemical analysis also showed that in polarized epithelial cells, e.g., renal tubular cells, epididymal principal cells or excretory duct cells, these calcium-binding proteins are present exclusively or mostly in the luminal plasma membrane.
Summary:For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e. g. by removal of serum albumin.Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives.We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-^step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigenantibody complex is eluted from the column.
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