del Rosario R B, Baron L A, Lawton R G, Wahl R L
Department of Chemistry, University of Michigan, Medical Center, Ann Arbor 48109-0028.
Int J Rad Appl Instrum B. 1992 Apr;19(3):417-21. doi: 10.1016/0883-2897(92)90128-l.
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.
