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 microns, covered by a silane coat carrying amino groups (Bio-Mag, 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 (micrograms/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 greater than IgG2b greater than IgG2a = IgG3 greater 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 greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hermentin
P
P
Research Laboratories of Behringwerke AG, Marburg/Lahn, Germany.
Doenges
R
R
Franssen
U
U
Bieva
C
C
Vander Brugghen
F J
FJ
Stryckmans
P
P
Friesen
H J
HJ
Optaczy
B
B
Schneider
S
S
Ax
W
W
D000911:Antibodies, Monoclonal / Q000276:immunology; D000943:Antigens, Differentiation / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D016827:CD8 Antigens; D002469:Cell Separation / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008280:Magnetics; D008863:Microspheres; D015260:Neprilysin; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D016827:CD8 Antigens; D005290:Ferric Compounds; D013438:Sulfhydryl Compounds; C000499:ferric oxide; D015260:Neprilysin