Antigens immunoaffinity

Antibodies and Antigens (Immunoaffinity Chromatography) [199], [200]. Immunoaffinity chromatography refers to the immobilization of antibodies (monoclonal or polyclonal) or antigens on an insoluble carrier (immunosorbent) for isolation and purification of the corresponding partner. 

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... 

Jasmer, D.P., Perryman, L.P., Conder, G.A., Crow, S. and McGuire, T.C. (1993) Protective immunity to Haemonchus contortus induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epitope. Journal of Immunology 151, 5450-5460. 

Another potential disadvantage of an immunoaffinity separation is the assumed abundance of the purified antigen in sufficient quantities to immobilize on a chromatography support. Protein antigens should be immobilized at densities of at least 2-3 mg/ml of affinity gel to produce supports of acceptable capacity for binding antibody. Often, the antigen is too expensive or scarce to obtain in the amounts needed. 

Figure 6.15 Principle of immunoaffinity chromatography. Only antigen that is specifically recognized by the immobilized antibody will be retained on the column...

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

Immunoaffinity chromatography makes use of immobilized antigen molecules to bind and separate specific antibody from a complex mixture. After the preparation of an... 

While the above-described sorbents are essentially nonspecific and designed to allow extraction of a wide range of analytes, there are also sorbent phases that are selective toward individual analytes, or at least classes of analytes. These are immunoaffinity (IA) sorbents and molecularly imprinted polymers (MIPs). In the first case, antibodies are immobilized on the solid support used for extraction, and the selective (in the ideal case specific) biochemical interactions allow an antigen to bind selectively to the antibody, whereas the other sample constituents are not retained and... 

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 continuing refinement in the selection of reference materials and the production of antibodies to complex protein mixtures has resulted in immunoassay systems of remarkable sensitivity and specificity. In particular, the selection and enrichment of the antibody population by immunoaffinity purification against the reference impurities has afforded an additional level of control over the production and validation of these reagents and served to improve the assay range and sensitivity (6,17). This normalization of the antibody population to a stoichiometric relationship with the reference impurities has suggested the term Antigen Selected Immunoassay (ASIA) for these methods. 

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