Anal. Chem. 1996, 68, 1646-1650
Homogeneous Bioluminescence Competitive Binding Assay for Folate Based on a Coupled Glucose-6-phosphate Dehydrogenase-Bacterial Luciferase Enzyme System Wei Huang, Agatha Feltus, Allan Witkowski,† and Sylvia Daunert*
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDHfolate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, doseresponse curves with a detection limit of 2.5 × 10-8 M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium. The high sensitivity associated with chemiluminescence and bioluminescence detection has been exploited recently in the design of analytical methods that have excellent detection limits.1-4 Among these methods, bioluminescence binding assays combine the sensitivity of bioluminescence detection with the selectivity provided by antibodies and binding proteins. Although several luciferases and photoproteins are potentially useful toward the development of bioluminescence binding assays, to date, the majority of the work has involved firefly luciferase,5,6 bacterial luciferase,7-9 and aequorin.10,11 Recent progress in the cloning of the aequorin and luciferase genes from several organisms has † Present address: BAS Analytics, 1205 Kent Ave., West Lafayette, IN 47906. (1) Stanley, P. E., Kricka, L. J., Eds. Bioluminescence and Chemiluminescence: Current Status; Wiley: Chichester, U.K., 1991. (2) Van Dyke, K., Van Dyke, R., Eds. Luminescence Immunoassay and Molecular Applications; CRC Press: Boca Raton, FL, 1990. (3) Campbell, K. Chemiluminescence; Ellis Horwood: Chichester, England, 1988. (4) Champiat, D.; Roux, A.; Lhomme, O.; Nosenzo, G. Cell. Biol. Toxicol. 1994, 10, 345-351. (5) Kobatake, E.; Iwai, T.; Ikariyama, Y.; Aizawa, M. Anal. Biochem. 1993, 208, 300-305. (6) Frayses, M.; Galen, F. X.; Habrioux, G. Eur. J. Clin. Biochem. 1992, 30, 433-437. (7) DeLuca, M. A., McElroy, W. D., Eds. Methods in Enzymology, Vol. 133; Academic Press: New York, 1986. (8) Terouanne, B.; Bencheick, M.; Balaguer, P.; Boussioux, A.; Nicolas, J. Anal. Biochem. 1989, 180, 43-49. (9) Balaguer, P.; Terouanne, B.; Eliaou, J.; Humbert, M.; Boussioux, A.; Nicolas, J. Anal. Biochem. 1989, 180, 50-54.
1646 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
provided a source of recombinant proteins that further enables the development of bioluminescence methods.12-14 Luciferase (from both firefly and bacteria) has been used in the development of several bioluminescence binding assays,5-9 all of which are of the heterogeneous type. In heterogeneous binding assays, one of the components of the system is typically immobilized on a solid support, and the assay requires a separation step. On the contrary, homogeneous assays do not require a separation step and are, therefore, easier to automate.15 We recently reported a homogeneous bioluminescence binding assay for biotin based on the inhibition of the bioluminescence of biotinylated aequorin by avidin.10 This assay had a detection limit of 1 × 10-14 M biotin, which is the best detection limit reported thus far for a homogeneous assay. The ability to detect such a low level of biotin was attributed to both the strong binding constant between avidin and biotin and the sensitivity associated with aequorin bioluminescence. Consequently, it is desirable to evaluate the feasibility of using other bioluminescent proteins, such as luciferase, in homogeneous competitive binding assays. In this study, we employed luciferase from the marine bacterium Photobacterium fischeri in the development of a homogeneous bioluminescence assay using folate as the model analyte. Folic acid is a vitamin that is required in the transfer of singlecarbon moieties, thus playing an important role in several metabolic pathways.16,17 These reactions are involved in the metabolism of amino acids such as serine, glycine, methionine, and histidine and the biosynthesis of the adenine, guanine, and thymine ring systems. Levels of folate in serum and red blood cells can be used in evaluating nutrition and in the diagnosis of folate deficiency, which may cause leukemia, megaloblastic anemia, and other diseases. (10) Witkowski, A.; Ramanathan, S.; Daunert, S. Anal. Chem. 1994, 66, 18371840. (11) Stults, N. L.; Stocks, N. F.; Rivera, H.; Gray, J.; McCann, R. O.; O’Kane, D.; Cummings, R. D.; Cormier, M. J.; Smith, D. F. Biochemistry 1992, 31, 14331442. (12) Meighen, E. A. Microbiol. Rev. 1991, 55, 123-142. (13) Prasher, D.; McCann, R. O.; Cormier, M. J. Biochem. Biophys. Res. Commun. 1985, 126, 1259-1268. (14) Inouye, S.; Aoyama, S.; Miyata, T.; Tsuji, F. I.; Sakaki, Y. J. Biochem. 1989, 105, 473-477. (15) Kasahara, Y. In Immunochemical Assays and Biosensor Technology for the 1990s; Nakamura, R. M., Kasahara, Y., Rechnitz, G. A., Eds.; American Society for Microbiology: Washington, DC, 1992; pp 169-182. (16) Stokstad, E. L. R. In Folic Acid, Proceedings of the Workshop; NAS: Washington, DC, 1975; pp 3-24. (17) Scott, J. M.; Weir, D. G.; Molloy, A.; McPartlin, J. Ciba Found. Symp. 1994, 181, 180-187. 0003-2700/96/0368-1646$12.00/0
© 1996 American Chemical Society
Luciferase from marine bacteria has found useful applications in bioluminescence assays for β-nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN), long-chain aldehydes, and related metabolites and enzymes.7 This luciferase utilizes NAD(P)H as the energy source and can be coupled to a variety of enzymes involved in the production or consumption of NAD(P)H. Among these enzymes are glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH), which have been used as enzyme labels in one of the most successful homogeneoustype binding assays, the enzyme-multiplied immunoassay technique (EMIT). Therefore, upon coupling the bioluminescence of luciferase with G6PDH (or MDH) that has been conjugated to a ligand of interest, the detection limits of the corresponding homogeneous assays should be significantly improved. A homogeneous enzyme-linked competitive binding assay for folate has been reported that used a single enzyme, glucose-6-phosphate dehydrogenase, as the label.18 Herein, we describe a bioluminescence assay for folate that couples the formation of NADH from the G6PDH enzymatic reaction with a bacterial luciferase bioluminescence reaction. In this assay, folate is attached covalently to G6PDH to form a G6PDH-folate conjugate, which can be inhibited by folate binding protein. The assay protocol involves competition between the G6PDH-folate conjugate and folate for a limited number of binding sites in folate binding protein. The inhibited activity of the conjugate is recovered proportionally to the concentration of the folate in the sample. Luciferase is responsible for generating the bioluminescence signal from the coupled G6PDH-luciferase enzyme reaction. Thus, the bioluminescence signal observed can be related to the concentration of folate in the sample, leading to assays with improved detection limits. EXPERIMENTAL SECTION Reagents. Luciferase and NAD(P)H:FMN-oxidoreductase from Photobacterium fischeri were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). N-Hydroxysuccinimide (NHS) was from Aldrich (Milwaukee, WI). Glucose-6phosphate dehydrogenase from Leuconostoc mesenteroides, folate binding protein (FBP) from bovine milk, β-lactoglobulin (containing FBP), folic acid, glucose-6-phosphate (G6P), β-nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN), ndecanal, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC), dithiothreitol (DTT), and all other reagents were from Sigma (St. Louis, MO) and were of the highest purity available. The following buffers and solutions were used: buffer A (0.0500 M Tris-HCl, 0.100 M NaCl, 0.010% (w/v) NaN3, pH 7.8), buffer B (0.100 M (NH4)2SO4, 0.100 mM DTT in buffer A), Tris-gelatin buffer (0.10% (w/v) gelatin in buffer A), β-lactoglobulin solution (0.20 mg/mL in buffer A), substrate solution (0.0100 M NAD, 0.0130 M G6P in buffer A), G6P solution (2.00 × 10-2 M G6P in buffer A), luminescence solution (1.20 × 10-4 M decanal, 2.00 × 10-5 M FMN, 2.00 × 10-3 M NAD in buffer A), luciferase/ oxidoreductase solution (10 µg/mL luciferase, 25 munits/mL oxidoreductase in buffer B). Luciferase and oxidoreductase were stored in buffer B at -20 °C. Apparatus. Bioluminescence measurements were performed with an Optocomp I luminometer from GEM Biomedical (Carrboro, NC) using a 100-µL fixed volume injector. The luminometer (18) Bachas, L. G.; Meyerhoff, M. E. Anal. Chem. 1986, 54, 956-961.
uses 75-mm × 12-mm glass tubes as cuvettes. A Syva (Palo Alto, CA) S-III spectrophotometer interfaced with a Syva CP-5000 PLUS clinical processor was used to determine the residual activity and maximum inhibition of each G6PDH conjugate. The spectra of G6PDH, folic acid, and the conjugates were obtained with a PerkinElmer (Norwalk, CT) Lambda 6 UV/vis spectrophotometer. Preparation and Characterization of G6PDH-Folate Conjugates. The G6PDH-folate conjugates were synthesized according to an N-hydroxysuccinimide/carbodiimide method.19 An amount of 100 units of G6PDH was used, and the product was diluted to a total volume of 2.00 mL after being dialyzed against buffer A. High initial folate/enzyme molar ratios were used to prepare highly substituted conjugates. The degree of conjugation was determined on the basis of the absorbance of the conjugates at 223 and 282 nm, as reported.20 The residual activity and maximum inhibition of the conjugates in the presence of binding protein were determined spectrophotometrically. For the determination of the maximum inhibition, a volume of 100 µL of a dilution of G6PDH-folate conjugate was incubated with 100 µL of β-lactoglobulin solution at room temperature for 20 min with shaking. Then, 300 µL of buffer A, 100 µL of Tris-gelatin buffer, and 600 µL of substrate solution were added. The absorbance at 340 nm was measured after a delay of 30 s, and the enzymatic activity was determined as the increase in the absorbance over a 1-min period. Study of the Kinetics of the Coupled Enzyme System. A volume of 100 µL of a dilution of G6PDH (or G6PDH-folate conjugate) in buffer A (buffer A for blank) was mixed with 200 µL of buffer A, 500 µL of luminescence solution, and 100 µL of luciferase/oxidoreductase solution, and the tube was placed in the luminometer. A volume of 100 µL of G6P solution was injected automatically with the built-in injector to trigger the coupled bioluminescence reactions, and a photon-counting program was started at the same time. The intensity of the bioluminescence signal was measured every 10 s as the total counts detected over 0.02-s periods and was expressed as counts per second. To study the kinetics, the bioluminescence intensity was monitored over a 5-min period. Determination of the Enzymatic Activity by Bioluminescence. Volumes of 500 µL of luminescence solution and 100 µL of luciferase/oxidoreductase solution were added to a test tube containing the enzyme sample, and the tube was placed in the luminometer. After the injection of 100 µL of the G6P solution to trigger the coupled reactions, the intensity of the luminescence was measured every 10 s as the total counts detected over periods of 0.1 s and was expressed as counts per second. The intensity at 60 s was used to determine the activity of G6PDH. The luminescence reagent and the luciferase/oxidoreductase solution were freshly made and kept on ice during each experiment. A calibration plot that relates intensity to G6PDH activity was constructed as follows. A stock solution of 2.5 units/L G6PDH was made in buffer A, and standard solutions were made from this stock. A volume of 100 µL of each of the standard solutions was mixed with 200 µL of buffer A, and the G6PDH activity was determined as described above. The detection limit for G6PDH was calculated from the signal that corresponds to two times the standard deviation of the background. (19) Bachas, L. G.; Lewis, P. F.; Meyerhoff, M. E. Anal. Chem. 1984, 56, 17231726. (20) DiTusa, M. R.; Schilt, A. A. J. Chem. Educ. 1985, 62, 541-542.
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Binder Dilution Curve. An amount of 1.0 mg of FBP was dissolved in 2.00 mL of buffer A to make a stock solution. Dilutions were made from this stock using Tris-gelatin buffer. Volumes of 100 µL each of the conjugate and FBP solutions were mixed and incubated at room temperature with shaking for 10 min. Then, 100 µL of buffer A was added, and the G6PDH activity was measured by bioluminescence as described above. The inhibition refers to the percent decrease in bioluminescence intensity relative to that in the absence of FBP. Dose-response Curve. Volumes of 100 µL each of the folate standard, diluted conjugate, and FBP solution were mixed and incubated at room temperature with shaking for 10 min. The resulting enzymatic activity of the mixture was determined as described above. Dose-response curves were prepared by plotting the percentage of inhibition of the enzymatic activity versus the logarithm of the concentration of folate in the standard solutions. Detection limits for folate were calculated from the signal that corresponds to two times the standard deviation of the background. Vitamin Tablet Sample Preparation. Two vitamin products from Basic Drugs (Vandalia, OH), vitamin for hair and multivitamin, were analyzed. Five tablets of each vitamin product were powdered and shaken vigorously with 5 mL of 1.0 M NaOH for 30 min. The mixture was centrifuged at 2500 rpm for 10 min, and the precipitate was discarded. The pH of the supernatant was adjusted to 7.0, and its volume was made to 100 mL with buffer A. Serial dilutions were prepared and analyzed according to the procedure described above. In addition, the cell growth medium RPMI-1640 (Sigma), containing a variety of vitamins, minerals, and nutrients, was analyzed after direct dilutions with buffer A. RESULTS AND DISCUSSION To develop enzyme-mediated homogeneous bioluminescence competitive binding assays, it is necessary to prepare conjugates between the analyte of interest and the enzyme label. These enzyme-analyte conjugates should retain sufficient enzymatic activity (i.e., have high residual activity), and this activity should be inhibited in the presence of an analyte-specific binder (e.g., antibody or binding protein). A series of G6PDH-folate conjugates with suitable inhibition and residual activity were prepared by varying the initial folate/G6PDH mole ratio used in the conjugation reaction. This initial mole ratio of folate to G6PDH determines the degree of conjugation of the G6PDH-folate conjugate, which in turn affects the residual activity of the conjugate and the maximum inhibition in the presence of FBP. Highly conjugated G6PDH tends to lose its activity. Therefore, it is necessary to optimize the initial folate/G6PDH ratio to produce the desired conjugate. From the prepared conjugates, the conjugate that had an initial folate/enzyme mole ratio of 600:1 produced a degree of conjugation of 29, was found to be inhibited up to 66% by FBP, and was used for all of the subsequent studies. Initially, a series of experiments were performed to determine the relative amounts of reagents (luciferase and FMN oxidoreductase) necessary to obtain optimum bioluminescence signal. Understanding of the light emission kinetics of the G6PDHluciferase coupled reaction system is important in order to be able to determine the activity of G6PDH by bioluminescence at low levels. Using a fixed concentration of NADH and in the absence of a dehydrogenase, Kurkijarvi et al. demonstrated that the kinetics of the bacterial luciferase reaction can be controlled by 1648 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
Figure 1. Typical light emission kinetics of the coupled bioluminescence reaction. The bioluminescence intensity was expressed as counts per second and was monitored every 10 s for 5 min ([, 1:2000 dilution of conjugate; 0, background).
the level of luciferase and oxidoreductase present in the system.21 They demonstrated that, for a fixed NADH concentration and in the presence of a low oxidoreductase activity, the rate of the luciferase-catalyzed reaction was constant, and a stable bioluminescence signal, which was directly proportional to the amount of NADH present, was observed. In the coupled reaction system employed in our studies, the NADH produced by the G6PDH enzymatic reaction results in an increase of the light intensity. This change in bioluminescence can be related to the activity of G6PDH, as will be shown below. It has been reported that partially purified bacterial luciferase contains different levels of FMN oxidoreductase, which affects the kinetics of the light emission.21 Furthermore, luciferase may be contaminated with dehydrogenase activity that could be responsible for causing a high background signal. Both of these undesirable effects can be avoided by using highly purified bacterial luciferase and FMN oxidoreductase. By employing such highly pure reagents, the kinetics of the light emission can be optimized so that the initial rate of the increase in bioluminescence corresponds linearly to the activity of G6PDH. Figure 1 shows a typical plot of the light emission kinetics of the G6PDH-folate conjugate, as well as one corresponding to the background (i.e., no G6PDH added). It was found that the light intensity measured at 1 min after triggering the luminescence reaction can be used to monitor the G6PDH activity without any significant loss of accuracy, and it was used in all subsequent measurements. As discussed earlier, because of the inherent sensitivity associated with bioluminescence detection, it was expected that our assay would yield a lower detection limit for G6PDH compared to those of the assays that employ spectrophotometric methods of detection. The calibration plot shown in Figure 2 indicates that the bioluminescence signal increases linearly with the activity of G6PDH at least up to 250 munits/L (equivalent to 250 µunits) and that the detection limit obtained was less than 1.5 munits/L (equivalent to 1.5 µunits) of G6PDH. For the rest of the (21) Kurkijarvi, K.; Raunio, R.; Lavi, J.; Lovgren, T. In Bioluminescence and Chemiluminescence: Instruments and Applications; Van Dyke, K., Ed.; CRC Press, Boca Raton, FL, 1985; pp 167-184.
Figure 2. Calibration plot for G6PDH. The concentration of G6PDH refers to that in the assays. Data points are means of triplicate measurements, and error bars denote ( standard deviations. Some error bars are obstructed by the symbols for the points.
Figure 3. Binder dilution curve of a 1:2000 dilution of conjugate. Data points are shown as an average ( 1 SD of triplicate measurements.
experiment, an amount of conjugate that produces a bioluminescence signal that is at least 30-fold higher than the background was used. This corresponds to about a 1:2000 dilution of the original G6PDH-folate conjugate preparation and is equivalent to 100 µunits of conjugate. A binder dilution curve (Figure 3) was constructed to determine the optimum amount of binding protein to be used in the assay. For that, a volume of 100 µL of a 1:2000 dilution of the conjugate was incubated with varying amounts of FBP. A plateau was reached at a high amount of FBP as all accessible folate on the conjugate was bound by FBP. An amount of binder that gives about 85% of the maximum inhibition is usually selected in the development of the homogeneous assay because it results in dose-response curves with excellent response characteristics.22 Since, under the assay conditions, the maximum inhibition of the (22) Kabakoff, D. S.; Greenwood, H. M. In Recent Advances in Clinical Biochemistry, Vol. 2; Alberti, K. G. M., Price, C. P., Eds.; Livingston C: London, 1981; pp 1-30.
Figure 4. Dose-response curves for folate (O, 1:1000 dilution of conjugate, 2 µg of FBP; 0, 1:2000 dilution of conjugate, 2 µg of FBP; 4, 1:1000 dilution of conjugate, 1 µg of FBP). Triplicate measurements were used for calculating the standard deviation. Concentration of folate refers to that in the standard.
conjugate by FBP is 60%, an amount of 2 µg of FBP was chosen to be used in the rest of the studies; this amount of FBP gives 50% inhibition of the conjugate. Dose-response curves for folate were constructed by incubating fixed amounts of G6PDH-folate conjugate and FBP with varying amounts of folate standards (Figure 4). This family of dose-response curves indicates the effects of FBP and conjugate concentration on the response characteristics of the assay. Two of the most frequently evaluated parameters in competitive binding assays are the detection limit and the ED50 value (ED50 is the concentration of analyte that corresponds to 50% of the maximum signal). When comparing the dose-response curves constructed by using 2 µg of FBP and varying dilutions of conjugate, it was found that when the lower conjugate concentration was employed, the dose-response curve was shifted toward lower (better) detection limits for folate and lower ED50 values. Specifically, for the 1:1000 and 1:2000 dilutions of the conjugate, the detection limits were 7.6 × 10-8 and 6.6 × 10-8 M, respectively. The corresponding ED50 values were 5.0 × 10-8 and 4.5 × 10-8 M, respectively. An additional comparison was made by using the same amount of conjugate (1:1000 dilution) and either 1 or 2 µg of FBP. The lower concentration of FBP gave a dose-response curve with an even better detection limit (i.e., 2.5 × 10-8 M in the standards, which corresponds to 2.5 pmol of folate) and a lower ED50 value (3.5 × 10-8 M). Thus, as the amount of FBP decreases, less folate is needed to effectively compete with the G6PDH-folate conjugate for the binding sites of FBP, resulting in an improvement of the detection limit and a lowering of the ED50 value of the assay. This is consistent with the observed shifts in the detection limits and ED50 values in Figure 4. Figure 4 also indicates that the location of the dose-response curve is determined by both the FBP and conjugate concentrations. The doseresponse curve obtained was steep over a narrow concentration range of folate, which leads to a sensitive assay for this analyte. Steep dose-response curves are commonly observed in homogeneous assays that use binding proteins rather than antibodAnalytical Chemistry, Vol. 68, No. 9, May 1, 1996
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ies.18,23,24 This is because the binding protein binds in a stronger fashion to the analyte than the enzyme-analyte conjugate due to steric reasons (i.e., steric hindrance in the case of the conjugate). This behavior is also consistent with a theoretical model of assays based on binding proteins.25 The developed assay was compared with a competitive binding assay for folate where the determination of G6PDH activity was performed by monitoring the rate of appearance of NADH at 340 nm (absorbance).18 The dose-response curve of the bioluminescence-based system yields assays with detection limits that are 1 order of magnitude better (i.e., 2.5 × 10-8 M folate). This is because the bioluminescence detection of G6PDH is more sensitive in comparison to the absorbance-based method. This allows for the use of a lower concentration of conjugate in the assay. Therefore, in our system, a lower concentration of FBP is needed to produce the inhibition of the G6PDH-folate conjugate. Consequently, a lower amount of folate analyte is needed to compete with the conjugate for the binding sites of FBP. Three different multivitamin preparations were analyzed for folate to validate this method with real samples. Two of the samples, multivitamin and vitamin for hair, were solid vitamin pills. The third sample was a liquid, cell medium RPMI-1640. The samples were pretreated as described in the Experimental Section and diluted to an appropriate concentration that would fit into the steep portion of the dose-response curve. The concentration of folate in each sample was extrapolated from the dose-response curve. As shown in Table 1, the homogeneous bioluminescence competitive binding assay for folate developed here yields values for folate in samples that match closely those reported by the manufacturer. It should be noted that the determination of the G6PDH-folate activity by monitoring the bioluminescence signal is compatible with high-throughput analysis. Multichannel luminometers are commercially available for use with microtiter plates that allow 96 samples on a plate to be measured at the same time. An added advantage to our method is that no conjugation between the analyte and luciferase is required. It has been reported that direct modification of luciferase is difficult due to the inactivation of the enzyme during the conjugation reactions.26 Moreover, DeLuca and Vellom attempted to develop a homogeneous assay for TNT by direct conjugation of luciferase.27 Although they were able to (23) Tsalta, C. D.; Bachas, L. G.; Daunert, S.; Meyerhoff, M. E. BioTechniques 1987, 5, 148-151. (24) Daunert, S.; Bachas, L. G.; Meyerhoff, M. E. Anal. Chim. Acta 1988, 208, 43-52. (25) Bachas, L. G.; Meyerhoff, M. E. Anal. Biochem. 1986, 156, 223-238. (26) Kricka, L. J. Clin. Chem. 1994, 40, 347-357. (27) DeLuca, M.; Vellom, D. NTIS Report; Order No. AD-A130090; National Technical Information Service: Springfield, VA, 1982. (28) Schroeder, H. R.; Vogelhut, P. O.; Carrico, R. J.; Buckler, R. T. Anal. Chem. 1976, 48, 1933-1937.
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Table 1. Results of Real Sample Analysis vitamin product
amount of folate founda
amount of folate claimed
multivitamin vitamin for hair RPMI-1640 medium
438 ( 14 µg/tablet 409 ( 27 µg/tablet 1.02 ( 0.05 mg/L
400 µg/tablet 400 µg/tablet 1 mg/L
a
Data shown as average ( 1 SD (n ) 3).
conjugate luciferase with a hapten, they were unable to inhibit the bioluminescence of the luciferase conjugate with a haptenspecific antibody; therefore, the development of a homogeneous luciferase-based assay was unsuccessful.27 Schroeder et al. have developed a competitive protein binding assay for biotin monitored by chemiluminescence. In that study, biotin was conjugated to isoluminol, and the chemiluminescence efficiency of this conjugate was enhanced upon the binding of avidin.28 In the assay reported herein, the bioluminescence is used only as a sensitive way to determine the activity of the enzyme label. Thus, coupling of an enzyme label that is easily conjugated and compatible with inhibition-based assays with a bioluminescence reaction can lead to homogeneous bioluminescence assays with improved detection limits. To the best of our knowledge, this is the first report of this type of homogeneous binding assay. In summary, we have demonstrated that the coupling of an enzyme label, such as G6PDH, with the bioluminescence detection produced from the luciferase system yields homogeneous binding assays with improved detection limits. In particular, a homogeneous bioluminescence binding assay for folate was developed. This assay was validated by determining folate in vitamin samples and culture media. Since there is a large number of available EMIT-type assays for numerous analytes that use G6PDH or MDH as the enzyme label, the coupling of these enzymes with luciferase could yield bioluminescence assays with improved detection limits for these analytes. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (GM 47915) and the National Science Foundation (CHE-9300384). A.W. and W.H. gratefully acknowledge the support of a Dissertation Year Fellowship and an Academic Year Fellowship, respectively, from the University of Kentucky. A.F. is an Otis A. Singletary Scholar at the University of Kentucky. Received for review July 31, 1995. Accepted December 21, 1995.X AC950757M X
Abstract published in Advance ACS Abstracts, March 15, 1996.
