New Ligand Coupling Procedure for Formation of an Immunoadsorption Wall

Abstract

To prevent the occurrence of dialysis-related amyloidosis, an immunoadsorption wall based on polyacrylamide has been manufactured by a recently developed, partially incomplete, two-stage polymerization method. During the preparation process, efficient utilization of coupling antibodies is the key to large-scale production of such a toxin removal modality. In this study, we attempted to carry out the ligand coupling procedure after formation of a cyanogen bromide (CNBr)-activated stationary phase, using anti-beta2-microglobulin (beta-2M) antibodies. In vitro immunoadsorption tests show that the levels of beta-2M decrease rapidly within the first 2 hours for all the immunoadsorption tests. After that, nearly blank values were reached for tests of initial levels of c. 30 microg/mL and c. 82 microg/mL, whereas a relatively constant level of c. 10 microg/mL was maintained for the test of initial levels of c. 185 microg/mL. The maximum surface binding capacity of the prepared immunoadsorption walls is estimated by fitting experimental data, using a mathematical model of saturation kinetics. The present comparative investigation also suggests the manufacturing process for an immunoadsorption wall could be improved and facilitated by this new ligand coupling procedure without compromising the resulting binding capacity. Furthermore, the experimental protocols as well as the present methodology could be helpful for development of a clinically applicable immunoadsorption wall.

In Vitro Removal of Beta‐2‐Microglobulin From Uremic Blood With an Immunoadsorption Wall

“…DRA, caused by the accumulation of β‐2M, remains a major concern in long‐term renal replacement therapies. In order to prevent or postpone the occurrence of DRA, we have attempted to manufacture an iWall aimed to serve as an adsorptive unit in conjunction with hemodialysis filters by a novel polymerization method and demonstrated its removal efficacy through simple experimental settings (21–25). However, there were several obstacles which impeded the progress of this technique.…”

“…The iWalls prepared by the partially incomplete two‐stage polymerization method were stored at 4°C in 0.15 M NaCl–10 mM sodium phosphate buffer (pH 7.4) (Sigma‐Aldrich, St Louis, MO, USA) containing 0.02% NaN 3 (Merck). Human β‐2M stock solution was fractionated from the urine of patients under hemodialysis and prepared as previously reported (24). β‐2M solutions with specified concentration levels for the present experimental studies were diluted from the stock early on the day of immunoadsorption tests.…”