Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.     

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.     

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

Synthesis of polyrotaxane-biotin conjugates and surface plasmon resonance analysis of streptavidin recognition

A polyrotaxane-biotin conjugate was synthesized and its interaction with streptavidin measured using surface plasmon resonance (SPR) detection. A biodegradable polyrotaxane in whichca. 22 molecules of α-cyclodextrins (α-CDs) were threaded onto a poly(ethylene oxide) chain (M _n: 4,000) capped with benzyloxycarbonyl-L-phenylalanine was conjugated with a biotin hydorazide and 2-aminoethanol after activating the hydroxyl groups of α-CDs in the polyrotaxane usingN,N′-carbony diimidazole. The results of the high-resolution¹H-nuclear magnetic resonance (¹H-NMR) spectra and gel permeation chromatography of the conjugate showed thatca. 11 biotin molecules were actually introduced to the polyrotaxane scaffold. An SPR analysis showed that the binding curves of the biotin molecules in the conjugate on the streptavidin-deposited surface changed in a concentration dependent manner, indicating that the biotin in the conjugate was actually recognized by streptavidin. The association equilibrium constant (K _a) of the interaction between the conjugate and streptavidin tetramer was of the order 10⁷. These results suggest that polyrotaxane is useful for scaffolds as a polymeric ligand in biomedical fields.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

Endogenous gibberellins and inhibitors in caryopses of rye

Gibberellins A₈, A₁₆, A₂₄, and abscisic acid were identified by GC-MS of derivatized extracts from immature fruits of Secale cereale. Mature caryopses contained ABA and trans-ABA in a ratio 1:1 as well as 4′-dihydrophaseic acid. During milk ripeness a neutral GA conjugate was detected. Free GA, afforded by enzymatic hydrolysis of the conjugate, was chromatographically identified as GA₁₆     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.     

Preparation and Characterization of Ligand-Modified Labelled Liposomes for Solid Phase Immunoassays

Abstract

Small unilamellar vesicles conjugated with an enzyme label and with specific ligands for biological molecules may prove to be useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other detection applications. Bifunctional vesicles have been prepared by covalently attaching horseradish peroxidase (HRP) and monoclonal antibodies to the outside of the lipid bilayer. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization with no apparent change in vesicle stability. These bifunctional vesicles were used in a noncompetitive immunoassay for D-Dimer, a fibrin dimer formed at the early stages of thrombogenesis. The assay results using vesicles led to a detection limit for D-Dimer in human plasma which was five times lower than what was achieved using a conventional enzyme-antibody conjugate assay. HRP labelled (bifunctional) liposomes can also be used in competitive assays for the detection of small ligands in bulk solution. HRP and biotin-conjugated vesicles were prepared and used in competitive assays for biotin in free solution. The lowest detection limit for biotin using vesicles as the signal generation mechanism was found to be a factor of 10 lower than what could be observed with a traditional biotin-HRP conjugate. A model has been developed for the competition between a small ligand in solution and a large ligand-conjugated vesicle for binding sites on a solid surface.     

Development of nonradioactive microtiter plate assays for nuclease activity

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Mechanistic investigation of smart polymer-protein conjugates

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

Enzymatic solid-phase assay for biotin and a biotin-benzodiazepine conjugate

A novel enzymatic ligand binding assay for biotin and its benzodiazepine conjugate is based on their binding to horseradish peroxidase-avidin conjugate (A-P) followed by the uptake of biotin-unsaturated A-P onto polystyrene beads coated with biotin-BSA. The detection limit is 1.3 x 10(-16) mol per tube (300 microL) with a 3.3 x 10(-12) M A-P solution and varies with the conjugate concentration employed. The coefficient of variation for 10 repetitive assays of 10(-15) mol of biotin is 6.22%.     

The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T₄) was prepared by N-acylation of N-(3-aminopropyl)biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T₄ conjugate with one or simultaneously with two binding proteins with affinity to Bt or T₄ in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T₄ was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T₄. The Bt-T₄ conjugate also formed a specific equimolar complex with T₄-binding human globulin (TBG) by the same mechanism as that for T₄. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T₄ conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T₄. The intact T₄ competitively inhibited this interaction after its addition to the system. Bt-T₄ also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu³⁺-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T₄ immobilized on a solid phase. Free T₄ inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T₄ complex was actively bound to the T₄-antibody, but had practically no interaction with serum T₄-binding proteins, including TBG. Probably, nonspecific interactions of the T₄ residue with streptavidin in its complex with Bt-T₄, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T₄-binding proteins of blood serum. The Bt-T₄ stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.     

Instability of the biotin-protein bond in human plasma

Labeling proteins with biotin offers an alternative to labeling with radioisotopes for pharmacokinetic studies in humans. However, stability of the biotin-protein bond is a critical tacit assumption. Using release of biotin from immunoglobulin G as the outcome, we individually evaluated stability of the biotin label produced by six biotinylation agents: biotin PEO-amine, 5-(biotinamido)-pentylamine, iodoacetyl-LC-biotin, NHS-LC-biotin, sulfo-NHS-LC-biotin, and biotin-LC-hydrazide. Each of the six biotinylated proteins was incubated at room temperature for 4h in human plasma or in phosphate-buffered saline (control). Free biotin was separated from the biotinylated protein by ultrafiltration and quantitated by avidin-binding assay. For each biotinylation reagent, biotin release was significantly increased by plasma (p < 0.0001 vs control by unpaired t test). Moreover, the hydrazide bond was also unstable in buffer. Biotin remaining on the protein was quantitated directly using capture of europium-streptavidin by the immobilized biotinylated immunoglobulin G. Consistent with biotin release data, streptavidin capture was reduced by plasma to 8% of control. We conclude that all of the biotinylating agents produce biotin-protein bonds that are susceptible to hydrolysis by factors present in human plasma; five of six are stable in buffer.     

Enzyme-linked coagulation assay: V. Amplified blotting assays using snake venom conjugates

We have previously reported an ultrasensitive microtiter plate assay, enzyme-linked coagulation assay (ELCA), which can measure a factor X activator isolated from Russell's viper venom (RVV-XA) at concentrations less than 0.1 amol/sample. The high sensitivity of this assay is derived from enzyme amplification via the clotting cascade in combination with the utilization of enzyme-labeled solution-phase and unlabeled solid-phase fibrinogen. Modification of the ELCA assay to detect RVV-XA directly bound to nitrocellulose, ELCA blot, as described in this report, allowed the detection of blotted RVV-XA at amounts as low as 2 fg. The high sensitivity of the ELCA blot was utilized to develop an immunodetection system for Western blots, the ELCA immunoblot, and a biotin/avidin protein stain for blotted membranes, biotin/avidin ELCA blot. For the ELCA immunoblot, using RVV-XA-labeled antibodies we were able to detect blotted placental alkaline phosphatase at amounts two orders of magnitude lower than those when using peroxidase-labeled antibodies. Using an avidin-RVV-XA conjugate in the biotin/avidin ELCA blot, 1 ng of biotinylated fibrinogen and 100 pg of biotinylated placental alkaline phosphatase, which had been subjected to electrophoresis and transferred to a nitrocellulose membrane, were visualized. These data support the general utility of the ELCA system for assay amplification on solid-phase matrices and demonstrate considerable potential of this methodology in "blotting" applications.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.