Synthesis And Characterization Of Adsorbents For Selectively Adsorbing Antibodies

Antibody is not only the main protein of the immune system that protects against disease by identifying and killing pathogens but also one of the pathogenic factors in autoimmune disease.Nowadays,apheresis using specific adsorbents is an effective method to remove IgG for clinical therapy of autoimmune disease.It has been reported that hydropobic ligands had certain abilities to selectively adsorb IgG.In this study,16 hydrophobic groups were researched including butan-1-amine,piperidine,phenylmethanamine,3-chloro-4-hydroxyphenylacetic acid,tyramine,dimethylaminoethylamine,dimethylaminopropylamine,methylpiperazine, benzene-1,3,5-tricarboxylic acid,4-(3-aminopropyl)morpholine,4-aminobenzensulfonamide, 4-(aminomethyl)pyridine,4-mercaptoetthylpyridine(4-MEP),4-amino-N-(6-methoxypyridazin -3-yl)benzenesulfonamide,indol acetic acid and thiomorpholine.They were coupled to agarose with three different activation methods and their dynamic binding capabilities(DBC) and selectivity under physiological conditions to IgG were researched. Some influential factors such as chemical structures of the ligands and the activation density of agarose have been studied.Experimental results were as follows:(1) The activation density of agarose had profound effect on its capacity to adsorb IgG. Results showed that the denser the activation density,the higher the IgG DBC.By analysing the tertiary structure of IgG,it was found that adsorbents can bind to IgG surface regions which are relatively flat and have many hydrophobic residues through multiple-point interactions.This kind of interaction was also stable under physiological conditions.So the increase of ligand density promotes the binding capacity of adsorbents to IgG.(2) Among 16 adsorbents,six adsorbents showed high IgG DBC(Q＞10 mg/mL gel). Indol acetic acid adsorbent had the highest(37.5 mg/mL gel) IgG DBC;next was 4-MEP adsorbent and its IgG DBC was 21.7 mg/ml gel in simple system.Neither adsorbents had high HSA DBC.(3) The adsorption constant(K_{a IgG},K_{a HSA}) of various adsorbents were assayed in pH 7.4, 0.01 mol/L PBS buffer under 37℃,using another main protein in huamn plasma:HSA as comparison.The selectivities of adsorbents were determined by K_{a IgG}/K_{a HSA}.Results showed that:phenylmethanamine adsorbent,butan-1-amine adsorbent,tyramine adsorbent, thiomorpholine adsorbent,3-chloro-4-hydroxyphenylacetie acid adsorbent,indol acetic acid adsorbent,4-MEP adsorbent had the capacities to selectively adsorb IgG(K_{a IgG}/K_{a SHA}＞10). (4) Based on the former research,six adsorbents showed high IgG DBC and selectivity in simply system.So their adsorption properties to human plasma were measured.Results showed that except 4-MEP adsorbent,all of the adsorbents had relatively low IgG sorption capacities in human plasma.IgG sorption capacity of indol acetic acid adsorbent decreased from 37.5 mg/ml gel to only 7.4 mg/ml gel.However,the sorption capacities of 4-MEP adsorbent to IgG,IgM and IgA were 25.6 mg/ml gel,1.1 mg/ml gel and 2.9 mg/ml gel, respectively.This work presented that 4-MEP adsorbent had high IgG sorption capacity and selectivity in human plasma.It shows excellent application prospect in the field of apheresis.