Antibodies released from immunoadsorbents: effect of support, activation and elution conditions.

Abstract

Immunoadsorption is an application of affinity chromatography, as a therapeutic method to specifically deplete biological fluids such as blood plasma from proteins in excess, or to extract a biomolecule from a complex mixture. However, the leakage of small amounts of antibodies covalently immobilized on the support hampers the practical use of this method. In fact, these released antibodies contaminate the purified proteins or depleted media and, when they are of animal nature, they may lead to immunization of patients, or cause an anaphylactic shock when a clinical use is concerned. It is therefore of prime importance that the immunoadsorbents exhibit a satisfactory stability over the whole range of chemical and biochemical conditions involved during their clinical handling. To determine optimal conditions for the preparation of stable immunoadsorbents designed to remove selectively Low Density Lipoproteins (LDLs) from the plasma of patients affected by familial hypercholesterolemia, various immunoadsorbents were prepared by covalent immobilization of goat anti-apolipoprotein B polyclonal antibodies on different supports (Sepharose CL-4B, Sepharose 6 Fast Flow, Sphérodex and Fractogel) previously activated by various chemical reagents (cyanogen bromide, divinyl sulphone, tresyl chloride and trichloro-s-triazine). Their adsorption capacity, specificity, stability and the amount of immobilized antibodies were compared in terms of the activation method and the support used. It turns out that the immunoadsorbents prepared with Sepharose 6 Fast Flow lead to optimal yield of coupling, adsorption capacity, and an excellent stability at neutral pH. TC-activated-Fractogel turns out as well to afford an excellent coupling yield, a good adsorption capacity and an optimal stability in the whole pH range tested.