Antibody Preparation

Either monoclonal or polyclonal antibodies may be used to prepare immunoaffinity columns. A variety of techniques may be used in the isolation of antibody from ascites, serum, cell medium, or other sources. Purification of antibody will reduce complications later in the analysis. Dependent on the properties of the antibody and the matrix from which it is isolated, ammonium sulfate precipitation (5), ion-exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, or affinity purifications using protein A or protein G are commonly used to purify the immunoglobulin (6). Protein concentration can be determined by UV-absorbance at 280 nm or colorimetrically using Bradford (7) or modified Lowry methods (8). The purity of the isolated immuno-globulin can be assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis based on the molecular weight of IgG (150,000 D).

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