Study On The Cleavage Of Anti-HCG By An Immobilized Papain Preparation And The Purification Of F?ab'?₂ Fragment

Human chorionic gonadotrophin is a glycoprotein,the synthesis and secretion of HCG is closely related to pregnancy.At present,complete antibody monoclonal antibodies are used for early pregnancy,but in the preparation of antibody which has higher specificity and sensitivity,The fragments which have smaller relative molecular mass shows more superior characteristics completed with intact IgG.That's because the relative molecular mass of F(ab')2 is two thirds of IgG's,and the removal of Fc produce a less resistance space for F(ab')2 fragments,it is available for the combination of antigen and antibody.Thus,papain was immobilized onto glutaraldehyde-activated paper cellulose under acid conditions.The enzymatic properties of the immobilized papain preparation were examined.Furthermore,the different methods were used to purify the F(ab')2 and the better methods to purify F(ab')2 fragments were established by comparison and parameters optimization,which laid the foundation for clinical application to rapidly diagnose and treat the patients,and provided a certain reference value to purification of other monoclonal antibodies.Some experimental results are as follows:1.The paper cellulose as carrier was activated by Epichlorohydrin and then the activated cellulose was aminated by ursol solution.The papain was then immobilized onto the glutaraldehyde-activated matrix.The immobilized papain has high activity,besides the enzymes and carrier combined firmly,it's not easy to fall off.And the immobilized papain can be reused.2.Ascites were purified by CA-AS and Protein A affinity chromatography,the former purity of the anti-HCG was about 90%,the latter purity of the anti-HCG was above 99%.3.The preparation of IgG fragments:The results indicated that fragments solution obtained by dissociative papain has impurities,including Fc fragments and dissociative papain,hydrolyze rate is 100%and purity is 74%.After purification,the purity can reach to 94%and the yield is 39%.4.The purity of IgG fragments:The results indicted that DEAE-Sepharose-FF chromatographic column was the suitable methods to apart IgG fragments,no matter gradient elution or stepwise elution,can apart IgG fragments from other hybridprotein.5.The purity and activity detection of IgG fragments:The results indicated that purity of F(ab')2 fragments were 74%which were obtained by dissociative papain and the other purity was 84%.The F(ab')2 fragments have activity,the titer was 1:12 000.