Antibodies separation methods

Owens SM, McBay AJ, Reisner HM, Perez-Reyes M (1981) 125 1 radioimmunoassay of delta-9-tetrahydrocannabinol in blood and plasma with a solid-phase second-antibody separation method. Clin Chem 27 619-624... 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

A more recent application of redox labeled ODNs is redox-active aptamers that exploit molecular recognition between the aptamer and a target analyte. Briefly, aptamers are functional nucleic acids that selectively bind to a variety of targets. Due to a well-defined three-dimensional structure, aptamers can achieve selectivity comparable to that of antibodies but are readily accessible taking advantage of well-known nucleic acid chemistry, polymeric chain reaction and contemporary separation methods, followed by aptamer selection from random pools of nucleic acids (DNA or RNA) by in vitro selection process called systematic evolution of ligands by... 

Ohlson, S., Nilsson, R., Niss, U., Kjellberg, B. M., and Freiburghaus, C. (1988) A novel approach to monoclonal antibody separation using high performance liquid affinity chromatography (HPLAC) with SelectiSpher-10 protein G. J. Immunol. Methods 114,175-180... 

Gee, A. P. (1998) Immunomagnetic cell separation using antibodies and superparamagnetic microspheres, in Cell Separation Methods and Applications (Recktenwald, D., and Radbruch, A., eds.), Marcel Dekker, Inc., New York, pp. 175. 

The first separation methods involved purification steps (chromatography or electrophoresis). Better manageable, but nowadays rarely found in commercial kits (however still used in research assays) are the methods in which the immune complexes are precipitated e.g. by addition of salts, organic solvents or a second antibody directed against the first antibody. All these techniques require a cumbersome centrifugation step. 

TABLE I Molecular Properties of Antibodies and Related Solid-Phase Separation Methods... 

Mammalian plasma from which polyclonal antibodies are extracted contain several hundreds of proteins, the most important in mass being albumin. Impurities constitute a difficult obstacle if classical separation methods are used however, affinity separation are efficient enough to eliminate most of foreign proteins. 

Figure 18 shows a separation of IgGl from a cell culture supernatant. As shown by electrophoresis, the purity of monoclonal antibodies in a single pass is of about 80-95%. This method represents the most advanced separation method for immunoglobulin for its good compromise between selectivity, binding capacity, and cost. 

TABLE 14 Summary of Chromatographic Separation Methods for Antibodies and Related Molecules... 

It is known that none of the already described methods for antibody separation provides sufficient pure, homogeneous products in a single chromatographic step. For higher purity, as required in therapeutic applications, the combination of two or more chromatographic procedures is mandatory. 

To achieve separation between Ag and Ag -Ab procedures that utilize differences in properties between the two components and maintain the Ag -Ab complex are used. If a separation method is ineffective, and/or markedly dissociates the complex, the erroneous conclusion can be made that antibodies are unsuitable for use in radioimmunoassays. Problems relating to the efficiency of separation procedures and those arising from contributions made to the blank values are discussed by Chard. ... 

Recently, we have also conducted experiments with antibody-based nanombe membranes for very high selective protein separations [47]. We found that immobilization of antibodies on the inner walls of the nanombes selectivity bind and transport its antigenic-protein molecules across the membranes. Remarkably, we have routinely observed selectivity coefficients as high as 34 for antibody-based membrane separation. In principle, antibodies can be raised for any molecules, and the antibody-tailored membranes can be used as a general but highly selective separation method. 

One of the most crucial considerations in proteomic analysis is sample preparation because this will ultimately dictate the number and type of proteins that can be processed. The first priority is to establish the precise protein system to be studied [e.g., will this be a comprehensive and exhaustive catalogue of every expressed protein within a tissue or cellular extract, or is only a small subset of a cellular proteome (e.g., only phosphoproteins or membrane-bound proteins) sufficient for analysis ]. Whether a full or partial proteome, or even a limited number of specific proteins is required for analysis, it is crucial that the extraction technique provide maximal protein recovery while preserving the integrity of the protein complex to be examined. Furthermore, the method of preparation must be totally compatible with the separation methods to be used. This is particularly important for separation technologies that are reliant on protein-protein interactions or drug/ligand/antibody, etc. interactions. Poor recovery of proteins is clearly... 

Affinity chromatography [14] is a highly-specific separation method based on biochemical interactions such as between antigen and antibody. The specificity of the interaction is due to both spatial and electrostatic effects. One component of the interactive pair, the ligand, is chemically bonded to a solid support, while the other,... 

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