Use of the immunosorbent

The sample is best applied to the immunosorbent in batch by rotating end-over-end for approximately 2 hours. Washing and elution of the desired material is most conveniently done by pouring the gel into a column. The best general eluant is 1 m acetic acid (pH2.3) applied with sufficient forward linear flow to elute the column over 0.5-3 hours. Elution of ligand in a batch process is sometimes ineffective. Alternative methods of immunosorbent elution use alkaline buffers (pH 11) or chaotropic agents such as 3 m potassium thiocyanate.

Several factors limit the separations which can be achieved with immunoadsorbents. Although in theory a single substance will be isolated, immunoadsorbents are necessarily ion exchangers because of the charges on antibody protein and are also capable of hydrophobic interaction with unwanted components of the sample. These contaminants are released along with the desired antigen when the immunoadsorbent is exposed to denaturant. The separation achieved therefore depends on the concentration of the antigen of interest relative to contaminants prevailing before application to the immunoadsorbent. For example, specific antibodies isolated by immunoadsorbent from high-titer immune serum (which may contain 20% of the specific antibody) are nearly homogeneous, whereas immunoadsorbent preparations of nonabundant proteins from crude cell or tissue extracts are composed principally of contaminants. Thus, immunoadsorbents are useful as a single step in the preparation of abundant proteins, or helpful in conjunction with several other independent separation techniques in the multistep purification of nonabundant proteins. Antibodies may lose avidity and capacity for antigen after immobilization and this tends to accentuate the limitations noted.

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