Single-chain antibody fragment-based adsorbent for the extracorporeal removal of β₂-microglobulin

Background. Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of β₂-microglobulin (β₂-m). Removal of β₂-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to β₂-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution. Methods. An antihuman β₂-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent β₂-m in serum. Results. The displayed scFv possessed a nanomolar affinity (K_D = 0.008 ± 0.004 mg-β ₂-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 ± 0.01 mg β₂-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed β₂-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human β₂-m. Conclusion. Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.