Separatıon of IgG Subclasses with Dye Affınıty Composite Cryogels

Abstract

Immunoglobulin G (IgG) is an antibody isotype. IgG, 150 kDa, is the most common type of antibody comprising about 80% of the body’s total amount of antibodies. IgG has four subclasses. Human IgG is composed of four isotypic subclasses termed IgG1 , IgG2 , IgG3 and IgG4. One of the indications of IgG administration is to enhance resistance of the patient to infections. Most often, IgGs directed against bacterial pathogens belong to the IgG2 subclass (Hammarstrom and Smith, 1986) . Hence, in situations of bacterial infections, it would be advantageous to use enriched IgG2 preparations. At present, there is no established method for the preparation of IgG2 -enriched solutions suitable for human therapy. Dye-ligand affinity chromatography is one of the chromatographic techniques which is widely used in recent years. The advantages of reactive dyes as affinity ligands for protein purification; economical and widely available, ease of immobilization, avoids hazardous and toxic reagents in matrix activation, stable against biological and chemical attack, storage of adsorbent without loss of activity, reusable: with stands cleaning and sterlization, ease of scale up, high capacity, medium specificity. The aim of this study, purification subunits of IgG (immunoglobulin G) using dye affinity composite cryogels. Poly(hydroxyethyl methacrylate) PHEMA beads were synthesized 71-100 m sized and Reactive Red 120 (RR) and Reactive Green HE 4BD were attached to these beads. Then the RR-HEMA and RG-PHEMA beads were embedded into the cryogel membranes to obtained RR-PHEMA and RG-PHEMA embedded composite cryogel discs RR-CCD and RG-CCD columns. The high porosity of cryogels makes them appropriate candidates as the basis for such supermacroporous chromatographic materials. Owing to supermacroporosity and interconnected pore-structure, such a chromatographic matrix has a very low flow resistance. Microsphere embedding method is a useful improvement for the combining the supermacroporosity and low flow resistance properties of the cryogel with large surface area and the high adsorption capacity properties of the microspheres. That was aimed the purification of IgG subunits with composite cryogels. The preapared composite cryogels were characterized by surface area measurments (BET), Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR), elemental analysis, swelling tests. Methods of seperation of IgG were determined. The parameters such as pH, concentration, temperature, flow rate and ionic strenght were investigated on adsorption of HIgG onto composite discs. The maximum adsorption capacity were obtained as 239.8 and 170 mg/g for RR-CCD and RG-CCD columns respectively. The adsorption of IgG subclasses onto adsorbents were also studied from both commercial HIgG solution and human serum. The reusability of the columns were tested and the desorption ratios were obtained about % 90 for both of the columns.