Synthesis And Evaluation Of Anti-β₂M Antibody Adsorbents

β2M is the light chain of major histocompatibility complex (MHC) class Ⅰ. It is involved in mutual recognition between cells and induction of immune responses. However, β2M of patients with renal failure can’t be normally metabolized in vivo so that it will be accumulated in the blood to become toxins. There are two methods for the treatment of complications of high β2M hyperlipidemia, i.e. drug therapy and surgery, both of which can only repair the damage and relieve the feeling of pain and have great side effect. Blood purification therapy can decrease the β2M level in blood from the source so that this way is considered one of the most effective ways of β2M hyperlipidemia. However, the selectivity of these existing blood purification materials is too low so that a lot of other useful molecules of the human body could be lost at the same time.In this paper, the specific IgY is used as a ligand for the affinity adsorbent to incerase its affinity and stability.The adsorbent performance is also evaluated. While the anti-β2M bivalent nanobodies (VHH2) is also expressed and its conduct activity have proved to be feasibly applied to blood purification treatment and the detection of P2M hyperlipidemia.(1) The acquisition and purification of specific IgY, First, β2M solution was used to immunize hens, and the crude extract supernatant was separated from the yolk. Two different paths of Sepharose-β2M adsorbent were synthetic to affinity purify specific IgY. The affinity purification of the second adsorbent was better than the first, and the titer of antibody was increased80-fold than the antibody before affinity purification.(2) The synthesis and Evaluation of IgY adsorbents. The adsorbent I and II were mainly through two different synthetic paths to which were measured on β2M binding affinity constants7.06x106L/mol and1.84x106L/mol. After two adsorbents reused five times its β2M adsorption capacity remained stable at50μg/mL and35μg/mL above. And then,the two adsorbents were immersed12h in the solution of0.01M NaOH solution and1mM dilute HCl. The supernatant was determined by indirect ELISA. The A450values were substantially equal to the blank control, with no loss group and their β2M adsorption capacity remained stable. In addition, both adsorbents did not produce significant non-specific adsorption of other serum components. (3) The expressment and activity verification of bivalent anti-β2M VHH2. The recombinant plasmid pET23a-β2M was transformed competent cells BL21(DE3), activated, and induction to be expressed. Its protein molecular weight was about30kDa, and expressed in the form of inclusion bodies, which accounted for32%of the total protein expression.The refolding dilution of inclusion body performed highest efficiency of25%. The activity verification of soluble protein after refolding dilution shows that part of the refolded VHH2remained active. This result proved that VHH2can be applied to the synthesis of blood purification absorbents or detection reagents β2M hyperlipidemia.