Immunoaffinity adsorbent

Preparation of immunoaffinity adsorbents require reasonably pure antibodies and usually salt/solvent fractions of antisera [28,33] or affinity purified polyclo-nal/monoclonal antibodies [18,22] are employed. Antibody purification strategies have been reviewed extensively [53, 65] and do not fall with in the purview of this article. It is however of interest to point out of a novel strategy employing thermo-sensitive immunomicrospheres that appear highly effective in the large scale purification of antibody from the sera of immunized animals [66,67]. 

The extent to which the immunoaffinity column can be reused depends mainly on the nature of the analyzed samples as well as the stability of the antibody and the support. The most important step is to remove any of the material physically adsorbed to the antibody so that the column may be reused with reproducibility. All that it is required for a column to be reequilibrated is the passage of several volumes of the starting buffer. Table 20.6 presents an outline of commercial column protocols used during analysis of steroid and -agonist residues in urine. Most columns have been shown to last at least 100 runs, provided that the sample is properly defatted and does not contain solid particles (169). 

In immunoaffinity chromatography adsorption phase is performed in physiological conditions followed by a wash with high ionic strength buffers to eliminate nonspecifically adsorbed proteins. Elution of human IgG is obtained by a deforming buffer such as a 0.2 M glycine-HCl, pH 2.7. 

To purify lamins. Drosophila extracts prepared as described above should be supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lamins from these extracts should be batch adsorbed to antilamin/protein A/sepharose by incubation overnight at 4°C. The column should then be washed extensively with equilibration buffer and purified lamins recovered from the antilamin affinity column with elution buffer. After elution, the solution containing the lamins should be immediately neutralized with Na2HP04 added to a final concentration of 50 mM. Immunoaffinity-purified lamins should be aliquoted, frozen by immersion in liquid nitrogen, and stored at -70°C until use. 

Affinity chromatography is a separation procedure in which the protein to be separated is selectively adsorbed by an immobilized secondary molecule. Undoubtedly the most specific method used to purify membrane proteins at the moment is immunoaffinity chromatography. In this method, the specificity of an antibody directed against a protein is used as a tool in the purification. 

This section describes a modified electrochemical immunosensor where a boronic acid approach is employed for attaching antibodies onto the surfaces of assay devices. It is commonly observed that antibodies often adopt random orientations and therefore fail to display their original immunoaffinity toward their corresponding antigen when they are directly adsorbed onto sensing surfaces. 

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