| Blood purification is a clinical treatment method for removing endogenous and exogenous toxins in patients’blood through membrane separation or adsorption.It can effectively relieve symptoms and has good therapeutic effect for patients with acute poisoning,organ failure and other critical diseases.In recent years,studies in the field of critical care medicine have shown that interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）,as typical pro-inflammatory cytokines,play an important role in the occurrence and development of"cytokine storm".This over-inflammatory response is a key factor in the progression of cancer immunotherapy,COVID-19,and bacterial infection.Therefore,the rapid and efficient removal of IL-6 and TNF-αfrom blood circulation of patients by blood purification technology with great value in the field of critical care medicine.Aiming at the problems of poor target selectivity and low efficiency of the existing technology in clinical use,this paper developed a specific blood purification adsorption material based on bispecificity antibody.This strategy is based on the antigen-antibody affinity recognition effect,and the Camel single chain antibody fragments（also known as nano antibodies）that can recognize IL-6 and TNF-αrespectively are expressed in tandem to obtain a bispecific antibody that can recognize both antigens.A highly selective adsorption material was prepared with bispecific-nanoantibody as the affinity ligand to further verify the adsorption effect of the material on the target in the plasma system.The specific results are as follows:（1）Preparation of monovalent nano antibody and IL-6.Anti-IL-6 nano antibody and anti-TNF-αnano antibody were prepared by prokaryotic expression with the equilibrium dissociation constants of 2.1×10-11M and 3.5×10-10M,respectively,using recombinant gene expression technique.The active human IL-6 was prepared by prokaryotic expression and dilution refolding technique with the refolding rate was 21%.（2）Preparation and performance evaluation of monospecific adsorbents.The specific adsorbents for IL-6 and TNF-αwere prepared by amino coupling using the monovalent nanoantibody as the functional base and agarose gel microspheres as the matrix,respectively.The results of adsorption evaluation showed that both adsorbents could bind target antigen effectively.The saturated adsorption capacity of IL-6 adsorbent was 7.45 mg/m L,and the maximum removal rate of IL-6 could reach 96%under simulated blood conditions.The saturated adsorption capacity of TNF-αadsorbent was 2.22 mg/m L,and the TNF-αremoval rate reached 86%.（3）The expression of bispecific nano antibody and the preparation and evaluation of adsorbent.Bispecific nano antibody was prepared by prokaryotic tandem expression.The antigen-binding properties of the bispecific nano antibody were characterized by non-denatured polyacrylamide gel electrophoresis,Biacore intermolecular interaction analyzer and liquid chromatography.The results showed that the equilibrium dissociation constants of the prepared bispecific nano antibody against IL-6 and TNF-αwere 1.7×10-11M and 3.7×10-10M,respectively,which were similar to the monovalent antibody.The bispecific antibody can bind to two antigens at the same time without affecting each other.And the saturated capacity of the adsorbent prepared by covalently immobilized bispecific antibody against IL-6 and TNF-αwas6.9 mg/m L and 2.19 mg/m L,respectively.The dynamic adsorption results showed that the removal rate of IL-6 was 97%,and the removal rate of TNF-αwas more than 84%.In conclusion,this paper successfully prepared a highly selective blood purification adsorbent using bispecific nano-antibody as affinity ligand,which can realize the recognition and adsorption removal of IL-6 and TNF-αin complex plasma system.The blood purification adsorbent is expected to play a role in the intervention treatment of cytokine storm. |
