Isolation and purification of immunoglobulins from chicken eggs using thiophilic interaction chromatography.

We report on the use of thiophilic interaction chromatography for the purification of IgY from egg yolk. This procedure permits the purification to homogeneity of IgY in a single chromatographic step after ammonium sulfate fractication. This study also compares the use of an improved T-gel which has a higher capacity for immunoglobulin than the original T-gel, having a capacity in excess of 25 mg IgY/ml resin. The recovery from this procedure is close to 100%, providing a simple and efficient means for purifying IgY from egg yolk. We also determined that the amount of specific antibody present in egg yolk from an immunised chicken is around 1% of total IgY.

Structure of the IgG-binding ligand of the T-gel.

We examine the ligand requirements for the divinylsulphone (DVS) based T-gel to bind immunoglobulins. The original gel consisted of 2-mercaptoethanol coupled to a DVS activated support, with both the thioether and sulphone sulphurs thought necessary for protein binding. No differences in the capacity for human IgG were observed for a highly activated gel coupled with mercaptoethanol, or when the same activated gel was incubated at high pH to hydrolyse the majority of its reactive groups before the remainder were coupled with the thiol, indicating that the thioether S may be replaced with a hydroxyl O. Increasing the time of the DVS-activation results in gels with higher concentrations of immobilised sulphone but lower concentrations of active groups. The IgG capacities of the mercaptoethanol coupled gels were found to increase with the time of the activation reaction, which may be exploited to produce high capacity gels while minimising the concentration of DVS. Reducing the vinyl of the DVS-activated gel with borohydride was found to decrease the amount of protein binding, with residual binding being attributed to the presence of hydrolysed or cross-linked sulphones in the gel. Reacting the activated gels with amines decreased the capacity for IgG still further, suggesting that not only are these ligands unable to bind IgG, they also prevent its interacting with neighbouring sulphones, perhaps due to the small amount of positive charge they carry.

Ligand structure of the divinylsulfone-based T-gel.

The requirements for divinylsulfone (DVS)-based gels to act as thiophilic adsorbents, binding immunoglobulins in a salt-dependent manner have been examined. No differences in protein binding were observed for a DVS-activated gel reacted with mercaptoethanol (the T-gel), or for the same gel treated at high pH to hydrolyse the active groups and/or allow the formation of cross-links within the matrix, indicating that an O atom may be substituted for the thioether without affecting the thiophilic interactions. Extending the time of the activation reaction between DVS and the matrix results in increased amounts of sulfone attached to the gel, but decreased levels of active vinyl groups. When coupled to mercaptoethanol, these adsorbents bound more IgG than gels activated for shorter periods. This provides a convenient method to prepare thiophilic adsorbents of high capacity while minimising the amount of DVS used. The immobilised vinylsulfone must be linked to an electron donating atom for IgG to bind. When the vinyl was instead reduced with sodium borohydride, protein binding was decreased. No IgG bound to amine-coupled DVS-activated adsorbents, perhaps due to an overall positive charge on these gels at pH 7.4. The binding of human IgG to the adsorbents is dependent on ligand density, with little protein binding to gels having less than 16 mumol sulfone per ml. The binding increased with the ligand density above this level, with more than 25 mg IgG binding per ml to an adsorbent having 114 mumol sulfone per ml. The lack of binding at low ligand densities would be expected if the IgG must interact with two or more sulfone ligands to be retained on the adsorbent.