Preparation Of Immunosorbent Using Heterofunctional Supports To Immobilize Protein A And Study Of Its Adsorption Performance

Autoimmune diseases and organ transplant immunological rejection are one of the problems in the medical field.The use of protein A immunosorbent for direct removal of pathogenic factors in the blood is currently the best treatment.For this reason,it is extremely important to increase the binding ability of the adsorbent to immunoglobulin(IgG).The immobilization of the affinity ligand on the support is a critical step in the preparation of the affinity chromatography material,and an effective immobilization method and a suitable support can significantly improve the effectiveness and cost of the immunosorbent.In the preparation of protein A immunosorbent,rSpA stability was increased by multi-point covalent ligation using recombinant protein A(rSpA)as a ligand,while orientation immobilization immobilized rSpA activity.The functional group on the surface of the heterofunctional support interacts with protein A under different conditions,so that it has a wide range of application,and the prepared protein A immunosorbent has high adsorption performance.In this thesis,the heterofunctional support immobilized protein A and its adsorption properties were studied.The details are as follows:Recombinant protein A(rSpA)was covalently bound to glutaraldehyde heterofunctional agarose using a targeted immobilization strategy.After controlling the structural difference between the two supports by controlling the glutaraldehyde concentration,pH and reaction time,it was found that each primary amino group on the agarose support had one or two glutaraldehyde molecules.In addition,SWISSMODEL was used to model the 3D surface structure of the B domain of rSpA,and MemBrain calculated the surface accessibility(ASA)value of amino acids to explore the distribution of reactive and adsorptive groups.Dimer glutaraldehyde agarose(Aga@DG)appears to react more amino acids with rSpA than monomeric glutaraldehyde agarose(Aga@MG).Therefore,the leakage of rSpA at Aga@DG@rSpA when adsorbing IgG was 0.24 ng/mg,which was slightly lower than 0.36 ng/mg of Aga@MG@rSpA.However,Aga@MG is more suitable for the orientation immobilization of rSpA,which allows the prepared adsorbent to have a higher IgG binding capacity.When rSpA was immobilized on Aga@MG at low and high ionic strengths,the maximum IgG binding capacity fitted by the Langmuir model was 56.2 and 59.2 mg/g,respectively,which was about 34% higher than at medium ionic strength.Compared to Aga@DG,Aga@MG offers shorter spacer arms and lower reactivity,which helps to positionally immubilized rSpA by controlling ionic strength.Heterofunctional aminoepoxy support prepared by activation of epichlorohydrin and 1,4-butanediol diglycidyl ether in different spatial microenvironments: amino and epoxy support(AAE),amino-epoxy support(AEA)and amino cyclic ether support(ABA).Three kinds of vectors were immobilized with rSpA under the same conditions(5 mM boric acid buffer,pH 8)to prepare three immunosorbents AAE@rSpA,AEA@rSpA and ABA@rSpA.By analyzing the structural differences of the three adsorbents,the B domain reactivity of rSpA and the distribution of the adsorptive groups,it is known that the spatial microenvironment affects the ion adsorption efficiency and steric hindrance of the support and rSpA when the orientation is covalently immobilized.Compared with the three immunosorbents,the maximum hIgG binding capacity of AEA@rSpA and AAE@rSpA was similar,and the maximum binding capacity was 45.8 mg/g and 42.1 mg/g,which was much higher than 18.2 mg/g of ABA@rSpA.On the one hand,the high hIgG binding capacity of AEA@rSpA is due to the suitable microenvironment on the support,on the other hand,it reduces the steric hindrance between the support and rSpA,which contributes to the rSpA immobilization rate on the support.In conclusion,SWISS-MODEL and MemBrain were used to model and calculate ASA values for exploring the distribution of rSpA reactivity and adsorptive groups in this dissertation.The high-adsorption immunosorbent material is obtained by preparing a heterofunctional glutaraldehyde support and an amino-epoxy support to immobilize rSpA,which be used antibody purification and treatment of autoantibody-related diseases in future.