Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads

Poly(hydroxypropyl methacrylate) [poly(HPMA)] gel beads with an average size of 150-200 micro m were prepared by suspension polymerization of hydroxypropyl methacrylate (HPMA). The poly(HPMA) gel beads were characterized by swelling studies, surface area measurements, scanning electron microscopy (SEM) and elemental analysis. Poly(HPMA) gel beads had a specific surface area of 88.6 m(2)/g. The dye Reactive Green HE 4BD was chemically attached to yield dye-poly(HPMA) gel beads at an average concentration of 44.3 micro mol dye/g bead with a swelling ratio of 75%. These dye attached gel beads were used in the separation of immunoglobulin-G (IgG) through adsorption-elution studies. The non-specific adsorption of IgG on the poly(HPMA) gel beads was 0.5 mg/g. The attachment of Reactive Green HE 4BD significantly increased the adsorption of IgG up to 71 mg/g. The Langmuir adsorption model was found to be applicable in interpretation of data pertaining to the adsorption studies of IgG with Reactive Green HE 4BD attached to the poly(HPMA) gel beads. The adsorption of IgG was found to be optimal at pH 7.0. The adsorption of IgG was observed to decrease by about 76% as the NaCl concentration was increased from 0.001 to 0.1 M. The IgG adsorption capacity of the dye attached poly(HPMA) gel beads was determined for a commercially available IgG solution to be 4.2 mg/g for IgG(1), 64.5 mg/g for IgG(2), 7.1 mg/g for IgG(3) and 10.8 mg/g for IgG(4). The Reactive Green HE 4BD attached poly(HPMA) gel beads have a significant adsorption capacity for IgG(2). The quantity of adsorbed IgG(2) is three times higher than the quantity of the other subclasses, IgG(1), IgG(3) and IgG(4). A similar adsorption behaviour was observed when the albumin free human plasma was used. The quantity of adsorbed IgG(2) is higher than the quantity of the other subclasses, IgG(1), IgG(3) and IgG(4). Adsorption capacities for albumin free human plasma were obtained as 6.4 mg/g for IgG(1), 67.8 mg/g for IgG(2), 5.2 mg/g for IgG(3) and 8.6 mg/g for IgG(4). Significant amount of the adsorbed IgG (up to 95%) was eluted in 1h in the elution medium containing 2.0 M NaCl. Repeated adsorption/elution processes showed that these dye attached gel beads are suitable for IgG adsorption.