Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion.

Iron oxide particles of average size 0.5-1.5 mu m, covered by a silane coat carrying amino groups (BioMag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (mu g/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM > IgG2b > IgG2a = IgG3 > IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS > NHIA > 2-It > SPDP. The magnetically responsive magnetite.-antibody conjugates ("magnetobeads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VlL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mu mol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used. This might be due to conformational changes of the antibody after coupling to the particles and to the cell surface receptor that is recognized. Separate batches of GMBS- and NHIA-magnetobeads may prove to be useful in a purging technique for allogeneic and autologous bone marrow transplantation, where one of the objectives is the elimination of residual T- and leukemic cells, respectively, contaminating the graft.