Enzymatic Preparation And Purification Of Monoclonal Antibody Fragments Against β-hCG

Monoclonal antibodies against β-hCG have great value on the diagnosis of early pregnancy and diseases such as placental-site trophoblastic tumor.However,the molecular weight of the complete monoclonal antibody is large and the structure is complex,which lead to its poor tissue penetrability,and prone to rejection between species.Antibody fragmentation is helpful to improve its properties.Thus study of the preparation of monoclonal antibody fragments against β-hCG by immobilized papain has been invested.Preparation and properties of thermally sensitive immobilized papain,preparation and purification of antibody fragments were investigated.Firstly,thermally sensitive material poly(N-isopropylacrylamide)which was esterified by N-hydroxysuccinimide,was employed as a carrier for the immobilization of papain.The immobilized enzyme exhibited the highest activity 84.7 U/mg after immobilization for 6 h at pH 8.5 and 22℃,with enzyme loadings of 0.6 mg/mg carrier.The optimal pH and temperature of immobilized papain were 7.5 and 65℃,respectively,and the Michaelis constant(Km)was 2.72 mg/mL.The immobilized papain remained 86.6%of the activity after 24 thermocycles.After stored at 4℃for 60 d,the immobilized papain activity retained 71.5%.To compare with free papain,the pH,thermal,operational and storage stabilities of immobilized papain were obviously improved.Then the thermally sensitive immobilized papain was applied to the digestion of monoclonal antibodies against β-hCG.The digestive condition was optimized.It was found that the largest amount of F(ab’)2 fragments were obtained when the enzymatic conditions were as followed:temperature was 30℃,time was 6 h,pH was 7.0,the proportion of immobilized papain and antibody was 50:1(U/mg).Compared with the papain immobilized on cellulose,the hydrolysis capacity of the thermally sensitive immobilized papain was higher and it was more suitable for the enzymatic hydrolysis of the macromolecules,especially the preparation of antibody fragments.At last,the antibody fragments were purified by gel filtration chromatography,Protein L affinity chromatography and Protein A-Protein L affinity chromatography.The purity and yield of F(ab’)2 fragments were 80.1%and 51.2%respectively when the antibody fragments were purified by gel filtration chromatography.Purity was 82.7%and yield was 46.5%after Protein L affinity chromatography purification.With the method of Protein A-Protein L affinity chromatography,98.4%purity was achieved with yield of 38.3%.The biological activity of F(ab’)2 fragments purified by these three methods was good.This study established the technology foundation of the application of antibody fragments in biological and medical field to a certain degree,and provided a certain reference value to preparation and purification of other monoclonal antibody fragments.