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At least 19 recordsLinked to original sources
PubMedDate not supplied

Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins.

In this investigation, a comparison of wild type recombinant streptavidin (r-SAv) with two genetically engineered mutant r-SAv proteins was undertaken. The investigation also included a comparison of the r-SAv with two streptavidin (SAv) proteins from commercial sources. In vitro characterization of the SAv proteins was conducted by HPLC, SDS-PAGE, IEF, and electrospray mass spectral analyses. All SAv proteins studied appeared to be a single species by size exclusion chromatography (HPLC) and SDS-PAGE analyses, but multiple species were noted in the IEF and MS analyses. In vivo comparisons of the SAv proteins were accomplished with dual isotope-labeled SAv in athymic mice. In an initial experiment, tissue localization of r-[131I]SAv directly radiolabeled using chloramine-T was compared with r-SAv radiolabeled with the N-hydroxysuccinimidyl p-iodobenzoate conjugate ([125I]-PIB), a radioiodination reagent that has been shown to result in iodine-labeled proteins which are stable to in vivo deiodination. The data obtained indicated that there is little difference in the distribution (except kidney localization) when r-SAv labeled by the two methods. Data obtained from comparison of r-[131I]SAv with a disulfide-stabilized r-SAv mutant (r-SAv-H127C), a C-terminal cysteine-containing r-SAv mutant (r-[125I]SAv-S139C), and two 125I-labeled SAv proteins obtained from commercial sources indicated that their distributions were quite similar, except the kidney concentrations were generally lower than that of r-[131I]SAv. On the basis of the similar distributions of the SAv proteins studied, it appears that the r-SAv mutants may be interchanged for the (wild type) r-SAv in pretargeting studies. Further, the similarity of distributions with two commercially available SAv proteins suggests that the results obtained in our studies and those of other groups may be directly compared (with consideration of animal model, sacrifice time, etc.).

Animals↗
PubMed1995

Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules.

Two recombinant core streptavidins were designed and characterized to understand the role of the terminal sequences, present in naturally truncated core streptavidins, in the properties of streptavidin. One recombinant core streptavidin, Stv-25, has an amino acid sequence very similar to natural core streptavidins. The other recombinant molecule, Stv-13, has further truncation of the terminal residues and consists essentially of only the beta-barrel structure characteristic of streptavidin. These recombinant core streptavidins are tetrameric and bind four biotins/molecule, as does natural streptavidin. The solubility characteristics of Stv-13, determined by varying the concentration of ammonium sulfate or ethanol, were almost the same as those of Stv-25 and natural core streptavidin. However, Stv-13 showed an enhanced structural stability compared with Stv-25 and natural core streptavidin. For example, Stv-13 retained greater than 80% of its biotin binding ability after incubation in 6 M guanidine hydrochloride at pH 1.5, under which conditions, Stv-25 and natural core streptavidin retained only about 20% of their biotin binding ability. In addition, Stv-13 showed higher accessibility to biotinylated DNA than natural core streptavidin. Apparently, the terminal regions, present on the surface of natural core streptavidin, spatially hinder biotinylated macromolecules from approaching the biotin binding sites.

Amino Acid Sequence↗
PubMedDate not supplied

Streptavidin in antibody pretargeting. 2. Evaluation Of methods for decreasing localization of streptavidin to kidney while retaining its tumor binding capacity.

An investigation has been conducted to determine if the kidney localization of recombinant streptavidin can be decreased to improve its characteristics in pretargeting protocols. Three different methods of accomplishing this were evaluated. The first method, blocking kidney uptake with a preadministration of recombinant streptavidin in which biotin occupied all of the binding sites, was unsuccessful. In a second method, l-lysine administration was used to block kidney localization. This method worked well, decreasing the concentration to 29% of the unmodified amount at 8 h postinjection. However, this method suffered from a requirement for constant infusion of lysine during the period of observation. A third method, use of succinylated recombinant streptavidin, was found to be the best approach. Succinylation of streptavidin was readily accomplished with very good protein recovery. With the succinylated streptavidin, the kidney concentration was only 14% of that of nonmodified streptavidin at 4 h postinjection. While these results demonstrated that the concentration of streptavidin could be decreased in the kidney, it was important to assess whether the tumor colocalization of streptavidin with biotinylated antibody was affected under those conditions. As part of our continuing investigation of pretargeting, a new water-solubilized biotinidase-stabilized biotinylation reagent was prepared. Using that reagent in a pretargeting experiment, an equivalent quantity of succinylated recombinant streptavidin as biotinylated antibody Fab' was localized in a tumor xenograft model. In that experiment, the kidney concentration was decreased to less than 10% of that obtained with unmodified recombinant streptavidin at 24 h postinjection. The results of our investigation have demonstrated that succinylation of streptavidin improves its distribution characteristics for pretargeting applications. The fact that succinylated streptavidin has no specific tissue localization should allow its use as a carrier of radioactivity in "two-step" pretargeting protocols.

Amidohydrolases↗
PubMed1989

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays.

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.

Bacterial Proteins↗
PubMed1993

A streptavidin mutant containing a cysteine stretch that facilitates production of a variety of specific streptavidin conjugates.

The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites. A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity. Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer. Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry. In this manner, S-[14C]carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared. These conjugates contain almost twenty [14C]carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive. More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.

Amino Acid Sequence↗
PubMed1990

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea.

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.

Bacterial Proteins↗
PubMed1998

Increased streptavidin uptake in tumors pretargeted with biotinylated antibody using a conjugate of streptavidin-fab fragment.

Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody.

Animals↗
PubMed1990

Tissue distribution of avidin and streptavidin injected to mice. Effect of avidin carbohydrate, streptavidin truncation and exogenous biotin.

Radioionated avidin and streptavidin were characterized for their biodistribution and tissue association in Balb/c mice, in comparison to their interaction with cells in vitro. Binding of avidin to spleen and bone-marrow cells in vitro was up to 20-fold higher than that of streptavidin, but when tested in vivo avidin clearance from blood and tissues was considerably faster than that of streptavidin. Levels of avidin at 24 h after an intravenous injection were below 1% (of the injected dose/mass tissue) in most organs. Non-glycosylated avidin was similar in its biodistribution to native avidin. Native streptavidin exhibited higher and prolonged tissue association with 5-10% levels in lung, liver, spleen, kidney and blood, whereas its truncated form showed low tissue levels (1-3%) but a remarkably high affinity to the kidney (80%). Exogenous biotin did not affect streptavidin distribution in vivo but caused a 2-7-fold increase in the retention of avidin (but not non-glycodylated avidin) in some of the organs.

Animals↗
PubMed1999

Organ selective delivery using a tissue-directed streptavidin--biotin system: targeting 5-fluorouridine via TNP-streptavidin.

Trinitrophenyl (TNP) modification of streptavidin (St) resulted in high and prolonged accumulation in mouse liver following intravenous administration of radioiodinated TNP streptavidin (TNP-St). Uptake, which is correlated with increased TNP substitution, was first observed at 2-3 h, increased to 40-50% of injected dose/gram tissue (%/g) at 24 h and slowly declined later on. A low degree of accumulation (10%/g) was observed in the spleen. TNP substitution of other proteins such as bovine serum albumin (BSA) or ovalbumin (Ova) led to a transient short-term liver uptake. The enzyme-resistance property of streptavidin and its biotin binding sites render TNP-modified streptavidin a potential targeting vehicle to the liver. 5-Fluorouridine (FUR) was attached to high molecular weight carrier carboxymethyldextran (CMdex, derived from 40 kDa dextran) and the dextran FUR conjugate was charged with 2-4 biotinyl groups (in the form of biotinyl-diaminopropionyl-tyrosine, BDT) for complexing to TNP-St. Biodistribution monitoring of the BDT-CMdex-FUR ligand, radiolabeled at the tyrosyl residue of BDT and targeted via non-radiolabeled TNP-St, showed that ligand accumulation in the liver was similar to TNP-St itself. Liver targeting of FUR was demonstrated by trace-labeling FUR with its structural analog 5,6-[3H]uridine prior to conjugation to dextran hydrazide. Specific liver accumulation of [3H] radioactivity occurred following administration of the conjugate only when complexed to TNP-St. Hepatic levels of [3H] radioactivity were in the range of 25%/g or 35% per whole liver during a period of at least 8 h, as compared to the rapid elimination of free FUR+[3H]uridine (4%/g at 20 min). [3H]-drug radioactivity disappeared at a faster rate as compared to 125I-dextran radioactivity, suggesting that metabolic processes required to generate the 5,6-[3H]uracil-containing active metabolites took place.

Animals↗
PubMed1999

[Cloning of streptavidin gene from Streptomyces avidinii and its expression in Escherichia coli. Secretion of streptavidin by E. coli cells].

The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created. It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells. The degradation site of the leader peptide was detected. Upon treatment with the total fraction of proteases secreted by S. avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.

Cloning, Molecular↗
PubMed1993

Surprising lability of biotin-streptavidin bond during transcription of biotinylated DNA bound to paramagnetic streptavidin beads.

We investigated the use of immobilized DNA templates as substrates for bacteriophage RNA polymerases in order to develop a simple method for separating template DNA from synthesized RNA. Double-stranded DNA molecules with a T7 or T3 RNA polymerase promoter at one end and a single biotin moiety at the other end were attached to streptavidin-coated paramagnetic beads and used in transcription reactions. When the biotin was attached by a nucleotide base on the nontemplate strand, the DNA-bead complex was moderately stable and could be used for multiple rounds of RNA synthesis. However, when the biotin was attached through a phosphodiester bond on the template strand, the enzymatic activity of RNA polymerase reversibly dissociated up to 80% of biotinylated DNA from the streptavidin beads. Biotinylated DNA bound to streptavidin beads in this system with a binding constant on the order of 10(12) M-1. These results stress the need for careful evaluation of solid phase adaptations of standard solution reactions in molecular biology.

Bacterial Proteins↗
PubMed2002

Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality.

A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.

Adsorption↗
PubMed2006

Heterotetramers formed by an S-layer-streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development.

Based on the S-layer protein SbpA of Bacillus sphaericus CCM 2177, an S-layer-streptavidin fusion protein was constructed. After heterologous expression, isolation of the fusion protein, and refolding, functional heterotetramers were obtained that had retained the ability to recrystallize into the square-lattice structure on plain gold chips and on gold chips precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Monolayers generated by recrystallization of heterotetramers on plain gold chips or on gold chips precoated with thiolated SCWP were exploited for the binding of biotinylated oligonucleotides (30-mers). Hybridization experiments with complementary fluorescently labeled oligonucleotides carrying one mismatch or no mismatch (both 15-mers) were performed and evaluated with surface-plasmon-field-enhanced fluorescence spectroscopy. For surfaces generated by the recrystallization of heterotetramers on SCWP-coated gold chips, a detection limit of 1.57 pM could be determined, whereas for surfaces obtained by direct recrystallization of heterotetramers on plain gold chips, a detection limit of 8.2 pM was found. Measuring the association and dissociation processes of oligonucleotides carrying no mismatch led to a dissociation constant of K(D)=6.3 x 10(-10) m, whereas for oligonucleotides carrying one mismatch a dissociation constant of K(D)=7.9 x 10(-9) m was determined. This finding was confirmed by measuring the whole Langmuir isotherm, which resulted in a dissociation constant of K(D)=2.6 x 10(-8) m.

Biotinylation↗
PubMedDate not supplied

Streptavidin in antibody pretargeting. 3. Comparison of biotin binding and tissue localization of 1,2-cyclohexanedione and succinic anhydride modified recombinant streptavidin.

Recombinant streptavidin (rSAv) is of interest as a carrier of alpha-emitting radionuclides in pretargeting protocols for cancer therapy. Due to the inherently high kidney localization of rSAv, modification of this protein is required before it can be useful in pretargeting. Previous studies (Wilbur, D. S., Hamlin, D. K. et al. (1998) Bioconjugate Chem. 9, 322-330) have shown that succinylation of rSAv using succinic anhydride decreases the kidney localization appreciably. In continuing studies, the biotin binding characteristics and biodistribution in mice of rSAv modified by reaction with succinic anhydride (amine modification) or 1,2-cyclohexanedione (arginine modification) have been compared. Modification of rSAv was conducted using 5-50 mol equiv of succinic anhydride and 60-200 mol equiv of 1,2-cyclohexanedione. Most studies were conducted using rSAv modified with the highest quantities of reagents. Succinylation of rSAv did not alter binding with biotin derivatives, but a small increase in the biotin derivative dissociation rate was noted for arginine-modified rSAv. Amino acid analysis of 1,2-cyclohexanedione-treated rSAv indicated about 40% of the arginine residues, or an average of 1.6 residues per subunit, were modified, whereas none of the lysine residues were modified. IEF analyses showed that the pI of the arginine-modified rSAv was 5.3-6, whereas the pI for the succinylated rSAv was approximately 4. Electrospray mass spectral analyses indicated that one to three conjugates of 1,2-cyclohexanedione, and two to three conjugates of succinic anhydride, were obtained per subunit. Both modification reactions resulted in greatly decreasing the kidney localization of rSAv (normally 20-25% ID/g at 4, 24, and 48 h pi). However, the kidney concentration for the succinylated rSAv continued to decrease (5% ID/g to 1.5% ID/g) from 4 to 48 h pi, whereas the concentration (5% ID/g) remained constant over that period of time for the arginine-modified rSAv. In contrast to this, the liver concentration appeared to be slightly higher (3% ID/g vs 2% ID/g) at the later time points for the succinylated rSAv. When less than 50 mol equiv of succinic anhydride were employed in the modification of rSAv, a correlation between increasing kidney localization with decreasing equivalents reacted was observed. Although the differences in the two modified rSAv are not substantial, succinylated rSAv appears to have more favorable properties for pretargeting studies.

Animals↗
PubMed2006

Comparison of a tetravalent single-chain antibody-streptavidin fusion protein and an antibody-streptavidin chemical conjugate for pretargeted anti-CD20 radioimmunotherapy of B-cell lymphomas.

The efficacy of radioimmunotherapy (RIT) for patients with relapsed non-Hodgkin lymphoma (NHL) is limited by nonspecific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled anti-CD20 antibodies (Abs). Pretargeted RIT using a covalent conjugate of the 1F5 anti-CD20 Ab with streptavidin (SA) has been shown to augment the efficacy of RIT and decrease toxicity compared with a directly labeled 1F5 Ab. We have engineered a tetravalent singlechain 1F5 (scFv)4SA fusion protein and compared it to the 1F5-SA conjugate. Athymic mice bearing Ramos lymphoma xenografts received either the conjugate or fusion protein, followed 20 hours later by a biotin-N-acetyl-galactosamine clearing agent, followed 4 hours later by 111In-DOTA-biotin. After 24 hours, 11.4% +/- 2.1% of the injected dose of radionuclide was present per gram of tumor (% ID/g) using 1F5 (scFv)4SA compared with 10.8% +/- 2.5% ID/g with 1F5 Ab-SA. Superior tumor-to-normal organ ratios of radioactivity were consistently seen using the fusion protein compared with the chemical conjugate (eg, tumor-to-blood ratio > 65:1 after 48 hours with the fusion protein, but < 7:1 with the conjugate). More than 90% of lymphomabearing mice could be cured with minimal toxicity using either reagent followed by 1200 muCi (44.4 MBq) 90Y-DOTA-biotin.

Animals↗
PubMed2005

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization.

Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin-streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (Deltaf) and dissipation factor (DeltaD) changes, together with the SPR angle shifts (Deltatheta), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin-DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin-DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotin-containing surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin-DNA assembled on the less structured streptavidin films formed through amine coupling.

Base Sequence↗
PubMed2002

Reduction of streptavidin RYDS-mediated renal adhesion by site-directed mutagenesis.

Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention. This retention is mediated by an integrin-binding motif-RYDS-that shares homology to the more common RGDS. We generated a c-Strep molecule constituting amino acids 13-139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES. RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E. coli and purified on a 2-imminobiotin matrix. Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers. The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay. RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin. Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.

Amino Acid Sequence↗
PubMedDate not supplied

Boron-enriched streptavidin potentially useful as a component of boron carriers for neutron capture therapy of cancer.

A boron-enriched streptavidin has been prepared by chemical conjugation of a boron-rich compound, B(12)H(11)SH(2)(-) (BSH), to a genetically engineered streptavidin variant. The streptavidin variant used has 20 cysteine residues per molecule, derived from a C-terminal cysteine stretch consisting of five cysteine residues per subunit. Because natural streptavidin has no cysteine residues, the reactive sulfhydryl groups of the cysteine stretch serve as unique conjugation sites for sulfhydryl chemistry. BSH was conjugated irreversibly to the sulfhydryl groups of the streptavidin variant via a sulfhydryl-specific homobifunctional chemical cross-linker. Quantitative boron analysis indicates that the resulting streptavidin-BSH conjugate carries approximately 230 boron atoms/molecule. This indicates that the chemical conjugation of BSH to the streptavidin variant was highly specific and efficient because this method should allow the conjugation of a maximum of 240 boron atoms/streptavidin molecule. This boron-enriched streptavidin retained both full biotin-binding ability and tetrameric structure, suggesting that the conjugation of BSH has little, if any, effect on the fundamental properties of streptavidin. This boron-enriched streptavidin should be very useful as a component of targetable boron carriers for neutron capture therapy of cancer. For example, a monoclonal antibody against a tumor-associated antigen can be attached tightly to the boron-enriched streptavidin upon simple biotinylation, and the resulting conjugate could be used to target boron to tumor cells on which the tumor-associated antigen is overexpressed.

Animals↗