Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human β-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N′,N′-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H2O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption–desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity.

Research highlights
► LDL cholesterol is a risk factor in the development of coronary heart diseases.
► Antibodies against LDL are used for the selective extracorporeal removal of LDL.
► Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel.
► PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion resistance and viscous samples can be easily applied on them.
► Anti-LDL antibody immobilized through protein A was more efficient than randomly immobilized anti-LDL antibody in removing LDL.