sepharose and desthiobiotin

With increased clinical use of antibodies, long-term delivery strategies are needed to decrease injection frequency and improve health outcomes. A three-component drug-delivery system was developed for competitive affinity release of a streptavidin-antibody conjugate from agarose-desthiobiotin hydrogels via controlled dissolution of sparingly soluble biotin derivatives. The antibody conjugate was localized in the hydrogel through streptavidin-desthiobiotin complexation. Dissolution of sparingly soluble biotin derivatives disrupts streptavidin-desthiobiotin complexation for controlled release of the antibody conjugate. Release was tuned by altering the total biotin derivative concentration without further hydrogel or antibody modification. First-order tunable release of bioactive Avastin, a therapeutic anti-VEGF antibody, was demonstrated from a non-cytotoxic system for over 100 days.

Topics: Antibodies; Biotin; Delayed-Action Preparations; Drug Carriers; Humans; Hydrogels; Sepharose; Streptavidin

Methods to reversibly control the chemical environment of hydrogels have application in three-dimensional cell culture to study cell proliferation, migration and differentiation in environments more representative of in vivo environments. Herein, we have developed a method to temporally control the chemical environment of agarose hydrogels through non-covalent attachment of peptide motifs. Streptavidin-GRGDS conjugates were immobilized in desthiobiotin-modified agarose hydrogels through the desthiobiotin-streptavidin interaction (K

Topics: Alginates; Biotin; Cell Culture Techniques; Cell Differentiation; Cell Movement; Cell Proliferation; Culture Media; Fluorescent Dyes; Human Umbilical Vein Endothelial Cells; Humans; Hydrogels; Oligopeptides; Peptides; Sepharose; Streptavidin

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.

Topics: Animals; Avidin; Biotin; Carrier Proteins; Chromatography, Affinity; Humans; Immunoglobulin G; Indicators and Reagents; Ligands; Protein Binding; Rabbits; Recombinant Proteins; Sepharose; Streptavidin; Tubulin

The adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin, desthiobiotin, or iminobiotin was measured. Under conditions that allowed only a limited number of molecular pairs to interact, the force required to separate tip and bead was found to be quantized in integer multiples of 160 +/- 20 piconewtons for biotin and 85 +/- 15 piconewtons for iminobiotin. The measured force quanta are interpreted as the unbinding forces of individual molecular pairs.

Topics: Adhesiveness; Avidin; Biotin; Ligands; Microscopy; Microspheres; Receptors, Cell Surface; Sepharose