engleski

Impact of dissociation constant on immunoaffinity and affinity chromatography

Affinity chromatography is a powerful purification method based on specific interaction of an e.g. enzyme and substrate, receptor and ligand, or antibody and antigen (immunoaffinity chromatography). Advantages of immunoaffinity chromatography are that it can be used for any protein (or even virus) regardless of its function and a very high binding affinity of antigen and antibody enabling isolation of a highly pure interacting partner. Disadvantage of the immunoaffinity chromatography is that a specific competitive elution cannot be employed as is the case with affinity chromatography employing e.g. enzyme or a receptor. Also, high affinity antigen- antibody interactions can only be disrupted using extreme conditions such as low pH conditions, denaturating agents or chaotropic salts. These conditions affect all noncovalent interactions, not only intermolecular but also intramolecular, potentially causing damage to the immobilized protein and the protein to be isolated making immunoaffinity inappropriate for general use. We aimed to find elution conditions disrupting antigen-antibody interactions but under native conditions. For testing of these elution conditions we used two immunoaffinity columns. In both cases columns were prepared by binding of antibodies to epoxy-activated monolith column. One column was specific for ovalbumin and the other for mumps virus. For comparison we also used tested elution systems with commercial protein G column and immunoglobulin solution. Effectiveness of tested elution systems was found different in these three columns, but the trend was the same. This indicates that possibly the differences in dissociation constants i.e. interaction strength are reflected in the obtained results regarding effectiveness of tested elution systems. So we would like to determine dissociation constant of ovalbumin-anti-OVA-antibody and to compare it with dissociation constant of protein G- immunoglobulin couple. Also, we would like to determine dissociation constants of mumps and measles viruses with their respective antibodies to gain insight into these interactions also.

affinity chromatography ; immunoaffinity chromatography ; dissociation constant

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