Immunoadsorption: Strategies for Antigen Elution and Production of Reusable Adsorbents

Abstract

Immunoadsorption is a powerful and generalizable method for protein purification that exploits the fine specificity of antigen-antibody interactions. In spite of its potential utility, the more widespread process scale use of immunoadsorption has been limited by the high cost of the antibody and the lack of gentle elution schemes that completely preserve the activity of both the immunoadsorbent and the eluted product. In this report, we review common chemical elution strategies such as pH, ionic strength, chaotropic salts, denaturants, and organic solvents as well as physical techniques such as pressure, electrokinetics, and temperature. In general, selection of elution strategies has largely been an empirical art, balancing stability of the immunoadsorbent and the eluted product and efficiency. The limitations of the available choices demonstrate that more attention must be placed on the antibody. Techniques which assist in the identification or creation of new antibodies with improved binding properties and resistance to degradation, e.g., screening and/or rational protein engineering, are also discussed.

Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins

“…This is because suitable elution conditions must be developed that disrupt the binding interaction between the immobilized ligand and the fusion tag without causing any degradation or significant changes in conformation of the target protein, leading to denaturation. Protocols using low pH eluents containing high concentrations of salts or chaotropic chemicals frequently result in a loss of function of the eluted proteins (Hoffman et al, 1980;Schmidt and Skerra, 1993;Yarmush et al, 1992). Milder elution protocols such as the use of excess ligand (competitive elution) can be inefficient, resulting in poor yields of the target protein (Thiele and Fahrenhoz, 1994).…”

SPR identification of mild elution conditions for affinity purification of E6 oncoprotein, using a multivariate experimental design

“…The construction or selection of antibodies with defined binding characteristics has also been suggested as tools for avoiding the use of harsh solutions (Yarmush et al, 1992;Burgess and Thompson, 2002). For non-tagged proteins, standard elution strategies are applied in trial and error approaches (for review see Yarmush et al, 1992;Firer, 2001).…”

“…The construction or selection of antibodies with defined binding characteristics has also been suggested as tools for avoiding the use of harsh solutions (Yarmush et al, 1992;Burgess and Thompson, 2002). For non-tagged proteins, standard elution strategies are applied in trial and error approaches (for review see Yarmush et al, 1992;Firer, 2001). The choice of the elution solution is generally arbitrary, although a few studies describe ELISA (Firer, 2001) or SPR (Brigham-Burke and O'Shannessy, 1993) elution assays.…”

“…Multivariate analysis can be used first to screen factors generally used to dissociate antigen-antibody complexes. In addition to pH, ionic strength, and temperature, chosen here because they should be compatible with E6 stability in a narrow range, chaotropic salts, denaturants, or organic solvents may also be assayed (Yarmush et al, 1992;Firer, 2001). Typically, 10 factors can be screened using 12-16 multivariate conditions.…”

Application of sol-gel glass immunoadsorbers for the enrichment of polycyclic aromatic hydrocarbons (PAHs) from wet precipitation

“…[2][3][4][5] Because of its influence on the final properties of the adsorber such as surface area, binding capacity, non-specific binding, and stability, one of the important steps in the preparation of an immunoadsorber is the choice of an appropriate support matrix. 6 Very common is the immobilization of the antibodies on agarose, and lately various silica materials have also been shown to be very promising. In most cases the antibodies are fixed chemically to the matrix, which can interfere with the activity of the biomolecules.…”