Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation.
Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [approximately 2 biotins/IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [125I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200-300 kDa range. By contrast, direct mixing with [125I]streptavidin rapidly gave larger oligomers of much greater than 669 and approximately 440-669 kDa molecular mass, respectively. The biodistribution of the 200-300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440-669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.
del Rosario
R B
RB
Department of Chemistry, University of Michigan, Medical Center, Ann Arbor 48109-0028.
Baron
L A
LA
Lawton
R G
RG
Wahl
R L
RL
D001426:Bacterial Proteins / Q000138:chemical synthesis*; D001710:Biotin / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000138:chemical synthesis; D007074:Immunoglobulin G / Q000737:chemistry*; D000922:Immunotoxins / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis*; D019809:Streptavidin
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D007074:Immunoglobulin G; D000922:Immunotoxins; D011108:Polymers; D001710:Biotin; D019809:Streptavidin