Protein antigens immobilization procedure

Immunoaffinity chromatography can be used to purify protein antigens by immobilizing the relevant antibodies on an inert matrix such as polysaccharide beads. When exposed to a protein mixture, only the protein recognized by that antibody will bind to the beads and can be eluted later in pure or almost pure form. Cells bearing the antigen on their surface can also be purified using a similar procedure. 

The ligand has to be immobilized on a sensor chip surface by either chemical cross-linking or with an affinity interaction like biotin-streptavidin (sensor chip SA), nickel chelate-His-Tag (sensor chip NTA) or antibody-antigen (sensor chip CMS). To ensure the measurement of accurate data the immobilization procedure should not interfere with the ligand-analyte interaction. The most common immobilization procedure for proteins is chemical cross-linking, which can reduce the activity or affinity of the interaction due to the modification of the protein or by blocking the binding site. 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Certain orientations may make a specific site on the protein inaccessible to ligand, substrate, or antigen. For example, consider the adsorptive immobilization of a specific IgG for a solid-phase immunoassay. The procedure will be optimal if the Fab domains are free to bind antigen (Figs. 1 and 16). 

The fractionation and purification of deteriorated proteins is undoubtedly one of the least successful techniques. This is simply because all of the methods that have been developed, with very few exceptions, are directed toward purifying the undeteriorated protein. The methods available are usually based on some particular biochemical activity of the protein, usually enzyme activity, and sometimes an affinity column or affinity adsorbent could be used to separate the native protein from the deteriorated one. Quite often a good affinity adsorbent is unavailable. This procedure, however, does not always work properly even when an adsorbent is available, because the deteriorated protein may possess some activity or an affinity for the adsorbent even though it has lost its natural enzyme activity (see Figure 24). The antigen-antibody reaction can also be used by means of precipitation with antibodies against the native proteins or adsorption on the immobilized antibodies. But here again, the specific antibody must be available, and the deteriorated protein may retain so much affinity for the antibody that differential separations will be impractical in some cases. 

Different methods are used to immobilize covalently the proteins to HPLC supports. The coupling procedures imply various activation chemistries via epoxide-, diol-, or aldehyde-silica [51,52], Generally, the immobilized antibodies are randomly oriented. Dihydrazide-silica supports are used for an oriented immobilization of the antibody through its carbohydrate residues [53]. Another approach is to bind the antibody to protein A or protein G surfaces [17]. The antibody will bind through the Fc portion, leaving the antigen combining sites oriented away from the support. 

For the determination of binding properties of biotinylated proteins and conjugates, use flexible 96-wells microplates (Nunc). The general design of the method is similar to classical enzyme-linked immunosorbent assay (ELISA) and RIA methods. Use the described procedure for immobilization of streptavidin, antigen, biotinylated or nonmodified antibody, or catalase. 

Immobilized Cells. Cells can be immobilized in the walls of UF hollow fibers and can grow to tissue-like densities (106-107 cells /cm2) between the fibers. This is about 10 times higher than densities achieved in roller bottles the hollow fibers act like natural capillaries in carrying nutrients to the cells and in removing toxic wastes. Cultures can be maintained for months at a time. Products like interferon, monoclonal antibodies, antigens, viruses and hormones may be produced continuously with dramatic increases in yield. In addition, where proteins such as monoclonal antibodies are being produced, subsequent purification steps can be simplified because the product occurs in high concentrations with lower concentrations of serum components than is the case with conventional mouse ascites fluid or suspension culture procedures. 

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