engleski

Nonspecific native elution of proteins and mumps virus in immunoaffinity chromatography

Immunoaffinity chromatography enables specific purification of any antigen by the use of the antibody raised against it, but also requires quite harsh elution conditions which might affect not only the intermolecular but also the intramolecular interactions. Therefore, the aim of this work was to find conditions that disrupt antigen-antibody interaction without affecting protein structure and stability. We hypothesized that solution of amino acids that are readily found at the antigen-antibody interaction site could act as a competing agent at neutral pH and result in elution of the antigen from the immunoaffinity column. We tested this hypothesis using two immunoaffinity columns ; specific for ovalbumin and mumps virus, and also using protein G affinity column. Immunoaffinity columns were prepared by immobilization of antibodies to epoxy activated monolith columns. Testing of elution potential of amino acids was constrained by limited solubility of amino acids. Results showed that the most efficient elution solutions were those containing imidazole and arginine and their efficiency increases with increasing molarity. Elution efficiency in proteins immunoaffinity chromatography was up to 40 %, but was shown to be beneficial in comparison to standard low pH elution regarding polymer content since amino acid eluates were polymer free in contrast to low pH eluates. Elution efficiency of mumps virus was up to 68 % and separation of noninfective virus particles was achieved. Amino acid solutions at neutral pH show modest elution efficiencies but combined with their stabilizing effect provide new possibilities of immunoaffinity chromatography use in protein and virus purification. *M. Brgles, D. Sviben, D. Forčić, B. Halassy, J. Chromatogr., A (2016) doi: 10.1016/j.chroma.2016.04.022

Immunoaffinity chromatography ; Native elution ; Virus ; Monolith

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