Bioconjugate
Chemxhy JULY/AUGUST 1990 Volume 1, Number 4 0 Copyright 1990 by the American Chemical Society
Enzymatic Solid-Phase Assay for Biotin and a Biotin-Benzodiazepine Conjugate Toshifumi Takeuchi and Garry A. Rechnitz’ Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822. Received November 8, 1989
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-l6 mol per tube (300 pL) with a 3.3 X 10-l2M 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 % .
INTRODUCTION Biotin conjugates, used in conjunction with the binding protein avidin, have found widespread application for cytologic identification, amplified detection, affinity purification, and related disciplines (1) because of the extremely strong binding of biotin to avidin with a dissociation constant of 10-l6 M (2). If biotin is used as a label for a target compound, a highly sensitive and selective method of determination is necessary. Currently, there are two approaches available for the determination of biotin, either a bioassay (3, 4 ) or a binding assay (524). Recently, the binding assay has gained preference, because the procedure is easier than that of bioassays with no sacrifice in sensitivity. These binding assays are based upon the competition of free and labeled ligands for a limited number of biotin binding sites on avidin. Diverse binding assays have been proposed, including isotopic (5121, colorimetric (13-17), fluorometric (18-21), chemiluminescent (22), and potentiometric (23, 24) techniques. The binding assays can be classified into two categories, homogeneous and heterogeneous. Homogeneous assays are faster and easier to perform than heterogeneous assays because no bound/ free separation step is necessary. However, due to matrix effects, it is difficult to measure a very low concentration in a complicated sample by homogeneous assay. In practice, a heterogeneous enzymatic assay (16) has a detection limit 100-fold lower than the most sensitive homogeneous assay ( 2 1 ) . 1043-1~
Consequently, we employed a heterogeneous system incorporating enzymatic amplification in an effort to develop a highly sensitive method for the determination of biotin and its conjugates, especially benzodiazepine drugs. EXPERIMENTAL PROCEDURES Materials. Nonporous polystyrene beads ( l / 4 in.) were purchased from Pierce (Rockford, IL). Horseradish peroxidase-avidin D (A-P, 1.5 mole of peroxidase per mol of avidin)’ was obtained from Vector Laboratories (Burlingame, CA). Biotinamidocaproyl-bovine serum albumin (biotin-BSA, ca. 9.6 mol of biotin per mol of BSA), bovine serum albumin, fraction V (BSA), 3,3’,5,5’-tetramethylbenzidine (TMB), and biotinamidocaproic acid N-hydroxysuccinimide ester (BAC-NHS) were obtained from Sigma Chemicals (St. Louis, MO). Hydrogen peroxide was bought from Aldrich Chemicals (Milwaukee, WI). A ben-
* Abbreviations used are as follows: A-P, horseradish peroxidase-avidin D; biotin-BSA, biotinamidocaproyl-bovine serum albumin; BSA, bovine serum albumin, fraction V; TMB, 3,3’,5,5’tetramethylbenzidine;BAC-NHS, biotinamidocaproicacid N-hydroxysuccinimide ester; HPLC, high-performance liquid chromatography; PBS, 10 mM phosphate buffer containing 0.1 M sodium chloride (pH 7.4); PBS-B, PBS containing 0.1% BSA DMF, N,N-dimethylformamide;Ai, an initial enzyme activity of free A-P; A,, an enzyme activity of free A-P after an incubation period.
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Figure 1. Uptake of A-P by the biotin beads. The beads are incubated with 3.3 X 10-12 M A-P at 4 O C for 1-8 h, then the enzyme activity in solution is measured. The recovery is expressed as 100(Ai - A.)/Ai! where Ai is an initial enzyme activity and A. is an enzyme activity of free A-P after a given incubation period.
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Figure 2. Standard curves of biotin: ( 0 )using 3.3 X 10-11 M A-P; (0) using 3.3 X 10-12M A-P; B, enzyme activity on the bead; Bo,enzyme activity on the bead without biotin. biotin
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Figure 3. Schematic diagram of the binding assay: (A) the biotin bead is added to sample, and A-P is then added; (B) biotin is bound to A-P (the first stage); (C) immobilized biotin complexes biotin-unsaturated A-P (the second stage). zodiazepinedrug, 1 0 1 2 4 was provided by Shionogi & Co., Ltd. (Osaka, Japan). The biotin-1012-53 conjugate was prepared by mixing BAC-NHS and 1012-S (1.2:l molar ratio) in 50 mM phosphate buffer at pH 7.0 and incubating it overnight at 4 "C. The product was then purified by a reversed-phase HPLC with 30% (v/v) acetonitrile/50 mM ammonium acetate buffer at pH 4.5. Other chemicals were purchased from Fisher Scientific (Kent, WA) and used without further purification. Immobilization of Biotin onto Polystyrene Beads. Before the immobilization, the beads were washed sequentially with detergent (Alconox),methyl alcohol, and water by using an ultrasonic bath and stored in 10 mM phosphate buffer (pH 7.4) containing 0.1 M sodium chloride (PBS) at 4 "C. Fifty beads were soaked in 10 mL of 0.002% biotin-BSA PBS solution overnight at 4 "C. To eliminate protein-binding sites, the beads were then incubated with 1%BSA in PBS. The biotin-immobilized polystyrene beads prepared (biotin beads) were stored in PBS containing 0.1 5% BSA (PBS-B) and 0.1 % sodium azide at 4 "C. To determine an average amount of biotin-BSA per bead, three sets of five beads in 1 mL of 0.002% biotinBSA were incubated overnight at 4 "C. The concentration of biotin-BSA in the supernatant was determined by the Bio-Rad protein assay based on the Bradford method (25)
using biotin-BSA as the standard. The solution incubated without beads was also assayed. The amount of biotinBSA was calculated from t h e difference in t h e concentrations. Binding Assay Procedure. A sample (100 pL) and PBS-B (100 pL) were pipetted into a test tube (10 X 75 mm) followed by addition of one biotin bead per tube. A-P (10-10 or 10-l1 M, 100 pL) was then added and mixed immediately. The mixture was incubated overnight (ca. 16 h) at 4 "C with continuous shaking at 80 oscillations/ min by a Precision Model 25 shaker bath (Chicago, IL). After the bead was washed five times with PBS, the enzyme activity was measured. In order to prevent the adsorption of A-P onto the test tube wall during complexation with biotin, the incubation mixture contained 0.033% BSA. I t is empirically demonstrated that BSA does not affect the binding of biotin to A-P. Measurement of Peroxidase Activity. A bead was placed in TMB solution (600 pL), prepared by dissolving 10 mg of TMB in 1mL of DMF followed by mixing with 100 mL of 0.1 M sodium acetate buffer (pH 5.51, and the enzyme reaction was started by addition of 0.01 % H202 (200 pL). The mixture was incubated for 5 min (for 6.3 X 10-11-4.0 X 10-lO M biotin) or 30 min (for 6.3 X 10-131.6 X 10-10 M biotin and 1.2 X 10-13-1.2 X 10-9 M biotin1012-S) with shaking. After an incubation period, the reaction was then stopped by addition of 0.2 N sulfuric acid (200 pL) and the absorbance was measured at 450 nm with a Sequoia-Turner Model 340 spectrophotometer. The activity in solution was determined by taking a 100-pL aliquot of it and assaying it with the same procedure used for a bead. Time Course of A-P Uptake by the Biotin Bead. Mixtures of M A-P in PBS-B (100 pL) and PBS (200 pL) were incubated with one bead per mixture at 4 "C for 1-8 h with continuous shaking at 80 oscillations/min. The enzyme activity in solution (A,) was measured and the recovery of A-P with the biotin bead was calculated by 100(Ai - A,)/Ai, where Ai is the initial enzyme activity in the incubation mixture. RESULTS AND DISCUSSION
Uptake of A-P with the Biotin Bead. For the characterization of the biotin bead prepared, biotinBSA adsorbed on the polystyrene bead was determined, and experiments of A-P uptake by the bead were performed. T h e average amount of biotin-BSA immobilized was 0.81 f 0.06 pg per bead, corresponding to about mol of biotin per bead. Although all of the immobilized biotin would not be available for the binding of A-P, it is expected that biotin on the bead is in large excess compared to the amount of A-P in the assay system, which is between 10-14 and 10-15 mol. In order to estimate the availability of immobilized biotin, the biotin bead was incubated overnight at 4 "C with 33 nM A-P dissolved in PBS-B (300 pL). After the incubation, 44% of the initial peroxidase activity remained in the solution. Nonspecific binding was estimated to be 8% by incubating the bead with biotin-saturated A-P. Therefore, it is estimated that 48% of A-P initially added is specifically bound to the bead, corresponding to approximately 5 X 10-12 mol. This figure represents a lower limit because additional experiments demonstrate that the bead has not yet been saturated with A-P under these experimental conditions. In the concentration range of A-P used in the assay procedure (0-3.3 X M), the amount of A-P bound increased linearly with the concentration. Consequently,
Bioconjugate Chem., Vol. 1,
Assay for Biotin and Its Benzodiazepine Conjugate 0.3
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Log [biotin-101241,M Figure 4. Standard curves of the biotin-benzodiazepine conjugate. Each biotin-10124 conjugate was incubated overnight at 4 O C with 3.3 X lo-'* M A-P and the biotin bead, then the enzyme activity was measured on the bead ( 0 )and in the liquid phase (0).
it is concluded that a sufficient amount of biotin-BSA is immobilized onto the beads for the purpose of this assay. Various factors contribute to a decrease in the observed rate of A-P uptake by the biotin bead (Figure l A ) , in contrast to the intrinsic association rate of avidin with biotin (2). Because the bead is nonporous, internal diffusion is not a factor. In a comparison of unstirred and vigorously agitated (80 oscillations/min) samples, the initial uptake rate observed was only twice as fast as that for the agitated sample. No further increase in the rate of uptake was observed for shaking a t greater than 80 oscillation/min. This implies that external diffusion is not a significant factor in the observed slow rate of uptake by the biotin bead. Therefore, the slow overall rate observed most likely is due to factors associated with the intrinsic properties of the unstirred boundary layer, e.g. steric effects of BSA covalently bound to biotin and protein-protein interactions between BSA and A-P. Biotin Binding Assay. The standard curves for biand 3.3 X M A-P otin obtained by using 3.3 X are depicted in Figure 2. The detection limit was 1.3 X 10-'6 mol and the coefficient of variation of 10 replicate assays for 10-15 mol of biotin was 6.22%. The range of biotin to be determined could be varied by altering the concentration of A-P used in the assay. Nonspecific binding of A-P decreased linearly when the initial concentration of A-P added was reduced. It was negligible when the concentration was below M, i.e. the absorbance is less than 0.002 for the enzyme activity of nonspecifically bound A-P on the bead with an incubation period of 30 min. In the experiments of uptake described above, when the log concentration of free A-P vs incubation time was plotted, a linear relationship was obtained (Figure 1B). These experimental data show that the overall uptake rate observed is first-order with respect t o free A-P concentration under the experimental conditions employed and was independent of immobilized biotin. Comparison of biotin samples that were preincubated with A-P and those with no preincubation showed no significant difference in the results. Because of these results, it is likely t h a t t h e present binding assay involves two noncompetitive binding steps (Figure 3): a fast step involving the binding of aqueous biotin by A-P (Figure 3B) and a slow step involving the complexation of unsaturated A-P with the biotin bead (Figure 3C). Thus, even without a preincubation step of samples with A-P, the experimental procedure gave quite reasonable results. Bayer et al. reported a sensitive enzymatic method for biotin using immobilized streptavidin on biotinyl-BSA adsorbed microtiter plates and detection by a biotinalkaline phosphatase conjugate (16). For the detection of
approximately mol of biotin, they used 0.1 pg of s t r e p t a v i d i n per s a m p l e , which was a n a m o u n t approximately 1700 times greater than that utilized in our system. Their incubation time for color formation by the enzyme reaction was recommended to be 20-24 h for the lower end of the biotin concentration range. In contrast, we employed the labeled avidin, allowing the amount of binding protein to decrease. In addition, we set up the procedure to include a long incubation for the uptake of the conjugate and a short incubation for the enzyme reaction. This gave high sensitivity and minimized errors associated with enzyme incubation. Determination of Biotin-Benzodiazepine Conjugate. Our aim is to establish a sensitive method for the determination of biotin conjugate. In order to preliminarily demonstrate the applicability of the proposed method, a biotin-benzodiazepine conjugate (biotin-1012S) concentration was determined. The structure of the conjugate a n d t h e a b s o r b a n c e change w i t h t h e concentration of the conjugate are shown in Figure 4. Although the measurement of the activity in solution was easier than that on the bead because of no bead-washing step, the change was measureably smaller than that for the solid phase. For a sample with a low concentration, the measurement of the activity on the bead is preferable. In this assay, a colorimetric detection method was employed, so the detection limit was 1.3 X 10-l6 mol. If more sensitivity is desired, a substrate with a fluorescent product like 3-(4-hydroxyphenyl)propionic acid is preferable. In conclusion, a highly sensitive assay method has been developed for biotin and a biotinylated benzodiazepine without special equipment. This binding assay will be applicable to the determination of biotin-drug conjugates used as probes in complicated systems, such as benzodiazepine binding studies in conjunction with benzodiazepine receptor proteins. These experiments are currently in progress. ACKNOWLEDGMENT The financial support of NIH grant GM-25308 is gratefully acknowledged. We wish to thank Kentaro Hirai and Urara Katsuyama, Shionogi & Co., Ltd. for providing 1012-S. We also thank Glenn B. Martin, Hawaii Biosensor Laboratory, and Akira Saito, University of Delaware, for valuable discussions. LITERATURE CITED (1) Bayer, E. A., and Wilchek, M. (1980) The Use of the AvidinBiotin Complex as a Tool in Molecular Biology. Methods of Biochemical Analysis (D. Glick, Ed.) Vol. 26, pp 1-45, John Wiley & Sons, New York.
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Takeuchi and Rechnitz (15) Niedbala, R. S., Gergits, F., 111, and Schray, K. J. (1986) A Spectrophotometric Assay for Nanogram Quantities of Biotin and Avidin. J. Biochem. Biophys. Methods 13, 205210. (16) Bayer, E. A., Ben-Hur, H., and Wilchek, M. (1986) A Sensitive Enzyme Assay for Biotin, Avidin, and Streptavidin. Anal. Biochem. 154,367-370. (17) Daunert, S., and Bachas, L. G. (1988)Homogeneous EnzymeLinked Competitive Binding Assay for Biotin Based on the Avidin-Biotin Interaction. Anal. Chim. Acta 208, 43-52. (18) Lin, H. J., and Kirsch, J. F. (1977) A Sensitive Fluorometric Assay for Avidin and Biotin. Anal. Biochem. 81, 442446. (19) Al-Hakiem, M. H. H., Landon, J., Smith, D. S., and Nargessi, R. D. (1981) Fluorimetric Assays for Avidin and Biotin Based on Biotin-Induced Fluorescence Enhancement of Fluorescein-Labeled Avidin. Anal. Biochem. 116, 264-267. (20) Mock, D. M., Langford, G., Dubois, D., Criscimagna, N., and Horowitz, P. (1985) A Fluorometric Assay for the BiotinAvidin Interaction Based on Displacement of the Fluorescent Acid. Anal. BioProbe 2-Anilinonaphthalene-6-Sulfonic chem. 151, 178-181. (21) Schray, K. J., Artz, P. G., a n d Hevey, R. C. (1988) Determination of Avidin a n d Biotin by Fluorescence Polarization. Anal. Chem. 60, 853-855. (22) Williams, E. J., and Campbell, A. K. (1986) A homogeneous Assay for Biotin Based on Chemiluminescence Energy Transfer. Anal. Biochem. 155,249-255. (23) Gebauer, C. R., and Rechnitz, G. A. (1980) Ion Selective Electrode Estimation of Avidin and Biotin Using a Lysozyme Label. Anal. Biochem. 103,280-284. (24) Kjellstrom, T. L. and Bachas, L. G. (1989) Potentiometric Homogeneous Enzyme-Linked Competitive Binding Assay Using Adenosine Deaminase as the Label. Anal. Chem. 61, 1728-1732. (25) Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72,248254. Registry No. Biotin, 58-85-5; biotin-1012-S, 127709-42-6.
