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Anal. Chem. 1989, 6 1 , 1728-1732
(25) Bod~r,N. Ann. N . Y . Acad. SCi. 1888, 289-306. (26) Rand, K.; Bcdor, N.; EIKwssi, A.; Road, I.; Mlyake, A,; Houck, H.; Gildersleeve, N. J. Med. Vkol. 1988. 2 0 , 1-8. (27) EIKoussl, A.; Bodor, N. Drug Des. Delivery 1987, 1 , 275-283. (28) Miyake, A.; Bodor, N., submitted for publication in J. Heterocycl. Chem (29) Arpino, P. J.; Devant, 0. Analysis 1979, 7, 348-354. (30) Harrison, A. 0. Chemical Ionization Mass Spectrometry: CRC Press: Boca Raton, FL, 1983; pp 65-7 1. (31) Wilson, M. S.;McCioskey. J. A. J. Am. Chem. SOC. 1975, 97, 3436-3444. (32) McEwan, C. N. Mass Spechom. Rev. 1986, 5 , 521-547. (33) Vestal, M. L. Mass Specfrom. Rev. 1983, 2 , 447-480. (34) Butferlng, L.; Schmelzelsen-Redeker, G.; Rollgen, F. W. J. ChromatCQr. 1987, 394. 109-116. (35) Derrick, P. J. Fresenius' 2.Anal. Chem. 1866, 324, 486-491. (36) Ligon, W. V.; Dorn, S. B. Int. J. Mass Spectrom. Ion Processes 1984, 6 1 , 3113-3122.
(37) Naylor, S.:Findels, F. A.; Gibson, B. A.; Williams, D. H. J. Am. Chem. SOC.1986, 108, 6359-6362. (38) Bodor, N.; Kaminskl, J. J. J. Mol. Sfruct. 1988, 763, 315-330. (39) Bodor. N.; Brewster. M. E.; Kaminski, J. J. J. Am. Chem. Soc., in press. (40) Bodor, N.; Brewster, M. E.; Kaminski, J. J. Tetrahedron 1988, 44, 7601-7610. (41) Cooks, R . G.; Busch, K. L. Int. J. Mass Spectrom. Ion Phys. 1963, 53. 111-124.
.
RECEIVEDfor review October 28,1988. Accepted May 8, 1989. This work was supported by NIH Grant GM-27167 and presented in part a t the 36th Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June 5-10, 1988.
Potentiometric Homogeneous Enzyme-Linked Competitive Binding Assays Using Adenosine Deaminase as the Label Thea L. Kjellstrom and Leonidas G. Bachas*
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
cumvents the necessity of sample dilution required to reduce the background absorbance and, therefore, should result in assays with better detection limits. Although several repork, describe the use of electrochemical means of detection in homogeneous enzyme immunoassays (EIAs) ( 5 ) ,there is only limited information available concerning the use of ion-selective electrodes to measure enzyme activity in such assays. A homogeneous EIA using a carbon dioxide gas sensor to monitor the inhibition of chloroperoxidase conjugated to IgG by an anti-IgG antibody was reported by Fonong and Rechnitz (6). This assay, however, was for a high molecular weight biomolecule. In another EIA, potentiometry was used to monitor the lysis of Micrococcus lysodeikticus cells loaded with trimethylphenylammonium ions by a lysozyme-biotin conjugate (7). Although quite elegant, this assay showed limited sensitivity; a total change in the signal of approximately 1.5 mV/min was obtained for a change in the concentration of biotin by 3 orders of magnitude. This report describes a homogeneous competitive binding assay for biotin based on the enzyme adenosine deaminase (ADA) and the strong and specific biotin-avidin interaction. In particular, binding of avidin to an ADA-biotin conjugate reduces the ability of the latter to deaminate adenosine. The competition between biotin in the sample and the ADA-labeled biotin for the avidin controls the enzymatic activity. The ammonia produced can be measured potentiometrically and can be directly related to the activity of the enzyme. Through dose-response curves the production of ammonia can also be related to the amount of biotin in the sample. These curves are extremely steep and therefore provide the basis for a highly sensitive assay for biotin. It should be mentioned that ADA has been proposed as an enzyme suitable for heterogeneous EIAs (8,9). However, this is the first report of using this enzyme for the development of homogeneous assays. Other heterogeneous electrode-based EIAs have been reviewed recently by Monroe (IO). EXPERIMENTAL SECTION Apparatus. For the static system studies, an Orion ammonia gas-sensing electrode (Model 95-12) was used. Voltages were measured with an Accumet pH/mV meter, Model 825 MP (Fisher
Homogeneous enzyme-llnked cmpet#lve Mndlng assays for blotln are described that are based on the competltlon between an enzyme-blotln conbate and free blotin for a flxed number of blndlng sltes of avidin. Unllke conventional homogeneous enzyme Immunoassays, in this system the analyte (biotln) is labeled with adenosine deamlnase (ADA), an ammonia-produclng enzyme. Consequently, potentlometric rather than photometric methods can be used as means of detection. Several ADA-Moth conjugates were prepared and showed as high as 97% inhibition of the enzymatic activlty In the presence of avidin. Addition of free biotin reverses thls InhlbRIon In an amount proportional to the concentratlon of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 X lo4 M. Varylng the concentratlon of the reagents In the assay allowed the detection llmlt and worklng range to be altered to a desired value. The proposed method was applied In the determinatlon of biotin in a horse-feed supplement.
INTRODUCTION Conventional homogeneous enzyme immunoassays are based on the EMIT (enzyme-multiplied immunoassay technique) principle (I). In these assays, a ligand-specific antibody binds to an enzyme-labeled ligand (conjugate), modifying its enzymatic activity. Unlabeled ligand reduces the antibodyinduced modulation of the enzymatic activity by competing for the specific binder and, therefore, leaving the enzymelabeled ligand free to react with the substrate(s) (2-4). In the commercial EMIT assays the enzyme label used is a dehydrogenase (glucose-6-phosphate dehydrogenase or malate dehydrogenase), and the enzymatic activity is monitored by photometric detection of NADH a t 340 nm. An inherent limitation of assays based on photometric detection is the background absorbance due to the color and turbidity of physiological fluids. A detection system based on a potentiometric rather than a photometric principle cir0003-2700/89/0361-1728$01.50/0
G
1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
Scientific, Cincinnati, OH), interfaced with an Alphacom 1842 printer (Fisher). The automated ammonia detection system was set up according to the specifications of Fraticelli and Meyerhoff (11). The gas dialysis unit consisted of two matched dialysis plates fitted with a GORE-TEX poly(tetrafluoroethy1ene)gas-permeable membrane (0.2-pm pore size) (W. L. Gore and Associates, Elkton, MD) (12). The flow-through electrode unit was constructed with a poly(viny1 chloride) ammonium-selective membrane using nonactin as the ionophore (11). Enzymatic activity was measured potentiometrically with the pH/mV meter and recorded on a Fisher Recordall Series 5000 strip-chart recorder. Reagents. Poly(viny1chloride), tetrahydrofuran, and dibutyl sebacate were from Polysciences, Inc. (Warrington, PA), Fisher, and Sigma Chemical Co. (St. Louis, MO), respectively. N Hydroxysuccinimidobiotin(NHS-biotin),ADA from calf intestinal mucosa, bovine serum albumin (BSA), and all other chemicals were purchased from Sigma. Deionized distilled water was used to prepare all solutions. Standard NH4+solutions ranging from 1.00 X to 1.00 X lo4 M were prepared from reagent grade ammonium chloride for the calibration of the detection systems. Calibration of the Orion electrode was performed in a 0.0100 M Tris-HC1 buffer, pH 8.5; a 0.100 M citrate buffer, pH 5.5, was used to recondition the electrode (13) between measurements. The buffers prepared for the flowing internal electrolytestream and the sample diluent stream of the automated system were 0.0100 M Tris-HC1, pH 7.5 and 8.5, respectively. The protein buffer consisted of 0.10% (w/v) protein (dialyzed gelatin or BSA) in assay buffer (0.0100 M M ethylenediaminetetraacetic Tris-sulfate containing 6.5 X acid (EDTA)). Both the assay and protein buffers were at either pH 8.5 (static system) or pH 7.4 (automated system). Substrate solutions were prepared by dissolving adenosine in the respective assay buffer for each system. All avidin and conjugate dilutions were made with protein buffer. Preparation and Evaluation of ADA-Biotin Conjugates. Adenosine deaminase was dialyzed in a 0.100 M sodium bicarbonate buffer, pH 8.2. Inosine was added to the enzyme solution in 3000-fold excess to protect the active site during conjugation. For each conjugation, 50 units of enzyme (at 4 "C) was used and enough volume of a NHS-biotin solution (10 mg of NHS-biotin in 1.0 mL of methyl sulfoxide) was added in aliquote to produce various biotin:enzyme ratios in the reaction mixture. Following dialysis against the assay buffer (pH 7.4), each conjugate was diluted to 2.0 mL with the assay buffer. The ADA-biotin conjugates were characterized with respect to enzymatic activity and inhibition by using the automated system. A volume of 100 pL of the appropriate conjugate was combined with either 200 pL of the protein buffer (activity) or 100 pL each of the protein buffer and avidin (inhibition). The mixture was incubated for 20 min prior to the addition of 1.00 M adenosine. The amount of ammonia produced mL of 4.4 X was measured in order to determine the activity and inhibition of the conjugates. Static System. A pH-dependence study was performed to determine the optimum pH at which the system as a whole (ADA-biotin conjugate coupled with the ammonia gas sensor) produced a maximum change in the millivolt signal. Tris-HC1 buffers were used for the pH range 7.5-9.0, and carbonate buffers were used for the pH range 9.5-10.0. To determine the effect of varying the incubation time, avidin (100 pL) was incubated for various time intervals with specific dilutions of the ADA-biotin conjugate (100 pL) and of the protein buffer (100 pL) in 2.60 mL of assay buffer. Adenosine (200 pL M solution) was added to the assay beaker, and of a 1.6 X after the appropriate incubation time, the ammonia produced was monitored for 5 min at 30-s intervals. A binder dilution curve was generated in order to determine the optimum binder concentration in the assay. One hundred microliters of different concentrations of avidin, 100 pL of ADA-biotin conjugate, 100 pL of protein buffer, and 2.60 mL of assay buffer were incubated together for 15 min; this time was also sufficient for the gas sensor to reach a stable base line. Then, 200 pL of 1.6 X M adenosine was added to the assay beaker, and the activity was measured as described above. The percent inhibition was calculated on the basis of the amount of ammonia
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produced in the presence of avidin relative to that amount produced in the absence of the binder. Dose-response curves were generated by using the same protocol as that described for the binder dilution study; however, a constant avidin concentration was maintained and 100 pL of the protein buffer was replaced with 100 pL of biotin standards. The percent inhibition was plotted against the logarithm of the concentration of biotin in the standards. Automated System. To investigate the association kinetics between the conjugate and the binder, 100 pL of an avidin solution (0.5 pg of avidin/mL) was incubated with 100 pL each of the ADA-biotin conjugate (1:200 dilution) and the protein buffer for set time intervals. After each incubation period, 1.00 mL of 6.5 x M adenosine was added and allowed to react for 15 min before stopping the reaction with 0.010 M silver nitrate. The ammonia produced was measured, and the percent inhibition was calculated and plotted against the incubation time. The effect of changing the amount of avidin on the percent inhibition of the conjugate was studied by incubating 100 pL each of the ADA-biotin conjugate (1:200 dilution) and protein buffer and varying amounts of avidin. One thousand microliters of 6.5 X M adenosine was added after a 20-min incubation period. The enzymatic activity was measured, and a binder dilution curve was constructed by plotting percent inhibition vs the amount of avidin used in each assay. The dose-response characteristics of the assay were studied by incubating a range of biotin standards with the ADA-biotin conjugate and avidin. One hundred microliters each of the conjugate, avidin, and biotin standard was incubated for 20 min M adenosine. prior to the addition of 1.00 mL of 6.5 X Doseresponse curves were prepared by plotting percent inhibition vs the logarithm of the biotin concentration in the standards. Feed Supplement Analysis. Biotin was extracted from a weighed sample (around 0.10 g) of a horsefeed supplement (Better Hoof from Horse Health Products, Louisville, KY) with 10.0 mL of 1.0 M NaOH. The pH of a 6.00-mL aliquot of this solution was adjusted to between 7 and 8. This solution was further diluted to 10.0 mL with the pH 7.4 assay buffer. The samples for the recovery studies were spiked with a known amount of biotin before extraction. Further dilutions of the supplement preparations and the spiked samples were made with the protein buffer, pH 7.4, so that they fit within the linear portion of the dose-response curve. The protocol for the analysis was that described for the dose-response study using the automated system.
RESULTS AND DISCUSSION Development of a precise and sensitive homogeneous enzyme-based competitive binding assay is dependent on the use of an enzyme-ligand conjugate that possesses high enzymatic activity and can be inhibited to a great extent. Several conjugates of ADA were prepared from the N-hydroxysuccinimide derivative of biotin (14),while the active site of the enzyme was protected by an excess of inosine. Such protection minimized the possibility of biotin attachment to lysines at the active site of the enzyme, which may result in a loss of enzymatic activity (15). In order to choose the best conjugate for the homogeneous competitive binding assay, each conjugate was evaluated with respect to percent inhibition, enzymatic activity, and percent residual activity (Table I). As expected, the least conjugated enzyme exhibited the greatest residual activity, 67 '70. Moreover, the residual activity of the conjugates decreased as the initial biotin:enzyme ratio increased. Contrary, increasing the initial biotin:enzyme ratio led to conjugates whose enzymatic activity can be inhibited by as much as 97% in the presence of avidin. Both of these observations may be attributed to a higher degree of conjugation (i.e., more biotins attached per enzyme molecule) when a higher initial biotin:enzyme ratio was used. Conjugate ADB4, with a 500:l initial mole ratio of biotin:ADA, showed 31% residual activity and 90% inhibition and was used for the remaining studies. Although ADA has been used in heterogeneous EIAs (8,9), this is the first time that a conjugate of this enzyme has
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Table I. Characterization of the ADA-Biotin Conjugates initial enzymatic residual biotin: activity: activity,b inhibition: conjugate enzyme ratio mU 9i 9i ADBl ADB2 ADB3 ADB4 ADB5
50 1 1oo:l 200:1
500:l 8W:I
67 64
28 58
3.2
51
1.9
31
1.5
25
72 90 97
4.2 4.0
"Milliunits, m u , refer to the enzymatic activity of 100 pL of ADA-biotin conjugate. (Unit definition: One unit of ADA deaminates 1.0 pmol of adenosine per minute at pH 7.4.) bPercent residual activity was calculated by comparing the activities of the conjugates to the initial activity of the unconjugated enzyme. 'Percent inhibition was determined by using k400 dilutions of the conjugates and an excess of avidin (20 pg). The incubation time was 20 min, in a total volume of 300 pL, after which 1.0 mL of 4.4 X M adenosine was added. The ammonia produced was monitored with an autoanalyzer system based on a flow-through ammonium-selective electrode. ~~
exhibited a reasonable degree of inhibition. Indeed, attempts to inhibit ADA, highly conjugated with adenosine 3',5'monophosphate (cyclic AMP), by an anticyclic AMP antibody were unsuccessful (16). Similarly, ADA-folate conjugates were inhibited by less than 10% in the presence of folate binding protein (17). A model is proposed here to explain all of the above observations. This model is based on the effect of the depth of the binding site of the various binders on enzyme inhibition. On the basis of electron microscopy studies, it has been reported that upon binding, the carboxylic group of biotin is buried about 9 8, below the surface of avidin (28). This distance is approximately 1.5 8, longer than the link provided by the side chain of lysine on the ADA-biotin conjugate. This allows for the avidin to reach the polypeptide backbone of the conjugate, thus forcing a change in the conformation of the enzyme. In comparison with avidin, antibodies have shallower binding pockets (18)and therefore are incapable of reaching the surface of the enzyme. It should be mentioned that conjugates of glucose 6phosphate dehydrogenase (GGPDH) with either biotin (14) or folate (19) can be inhibited up to 100% and 7070, respectively, in the presence of their corresponding binding proteins. Similarly, conjugates of this enzyme with other ligands such as therapeutic drugs, drugs of abuse, and hormones can be inhibited in the presence of ligand-specific binders (20). Therefore, it appears that it is easier to inhibit conjugates of G6PDH than of ADA. One possible reason for this is that unlike ADA, which needs only one substrate (adenosine), dehydrogenases catalyze redox reactions and require the presence of two substrates. This, along with the fact that NAD is larger than adenosine, implies that the region of the enzyme responsible for substrate recognition is larger in the case of G6PDH and thus more likely to be affected by the binder. It is customary to include a high concentration of a nonreactive protein in the assay mixtures to prevent nonspecific binding of the biomolecules to the walls of the assay container (21). Gelatin was chosen in our initial experiments; however, due to its high ammonia content, it was necessary to dialyze this protein against the assay buffer before use. It was later found that BSA was a more suitable buffer protein. The ammonia background signal due to the BSA buffer was appreciably less, thus eliminating the preassay dialysis step. It was also found that the nature of the protein itself had no effect on the response characteristics of the assay. In this study, two different types of detection systems were used to measure enzymatic activity. In our initial studies, an Orion ammonia gas-sensing electrode was employed. The
t
-21 7.0
I 8.0
I
I 9.0
I
I
I
io0
PH Flgure 1. Effect of pH on the enzymatic activity of ADB4 in the absence (0)and presence (0)of 1 pg of avidin. The y axis refers to the change in potential per minute due to the productkn of ammonia.
Data points are means of duplicate measurements.
6of
40
2oLl!LuAu 00
IO
20
30
40
50
60
Incubation Time, min Flgufe 2. Effect of varying the incubation time between the conjugate and avidin on percent i n h i b b (0)100 pL of a 1:lOO daution of ADB4 and 1 pg of avidin (static system); (0)100 pL of a 1:200 dilution of ADB4 and 0.5 pg of avidin (automated system). Values shown are averages of duplicate measurements.
optimum pH for ADA is 7.4; however, the Orion electrode has an optimal response at pH 9.5 (9). Therefore, in order to find the best conditions for our experiments, it was necessary to evaluate the electrode signal produced as a function of pH. Figure 1shows the results both in the absence (activity) and presence (inhibition) of avidin. From these data, the optimum pH was determined to be around 8.5. At this pH the electrode detected the greatest difference in the enzymatic rate between the activity and inhibition of the ADA-biotin conjugate. Therefore, controlling the p H a t 8.5 should result in more precise assays with better detection limits. A maximum inhibition of 84% was attained in the presence of avidin (the avidin and conjugate were incubated for 15 rnin). Although a conjugated enzyme (ADB4) was used in these studies, the optimum pH was similar to that reported in an earlier study using unmodified adenosine deaminase with the same detection system (9). To evaluate the association kinetics between the conjugate and avidin, a study was undertaken in which the incubation time was varied (Figure 2). A steep rise in percent inhibition was observed; after an incubation period of 5 min the conjugate was already inhibited by 60%. For the studies based on the ammonia gas sensor, a 15-min incubation period was selected that corresponded to 87 70 inhibition. Following optimization of the pH and the incubation time, the effect of changing the amount of avidin on the inhibition of a constant amount of ADB4 was evaluated. As shown in
ANALYTICAL CHEMISTRY, VOL. 61,NO. 15, AUGUST 1, 1989
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IO0 80 60
40
20
0 Avidin, pg Flgure 3. Effect of varying the levels of avidin (micrograms of avidin in the assay mlxture) on the lnhibitlon of a 1:lOO dilution of AD84 (static system). Means of duplicate measurements were plotted.
Figure 3, as the amount of binder increases, the inhibition increases. The least amount of binder that produces a measurable inhibition is around 0.1 r g (equivalent to 1.5 X lo4 M),which indicates that this method can be used for the sensitive determination of avidin. Further, this curve is useful for the selection of the appropriate concentration of avidin for the biotin assay. Typically, the amount of binder that corresponds to approximately 85% of the maximuminhibition is chosen in order to obtain doseresponse curves with the best detection capabilities (19,20). As shown in Figure 3, 2 pg of avidin produced an inhibition of 86%;however, 1pg of avidin resulted in an inhibition of 71% and was used for the doseresponse studies. For the latter, free biotin was incubated along with ADB4 and avidin in a single incubation mode of analysis (22)before addition of the substrate. Dose-response curves were obtained that related the concentration of biotin in standards to the inhibition of the enzyme-ligand conjugate (Figure 4). The EDw value (effective dose at 50% of maximum response) of this curve is 4 X lo-’ M. Unfortunately, the use of the commercial gas sensor limits the number of samples that can be analyzed to about three per hour. To circumvent this limitation, an automated gassensing detection system was used. The purpose of our initial study with this system was to evaluate the effect of varying the incubation time between avidin and ADB4 (Figure 2). The steepness of the slope between zero and 15 min of incubation time emphasizes the quick response capability of this assay. A value of 75% inhibition was observed at an incubation time as short as 5 min. For the remainder of the studies an incubation period of 20 min, corresponding to 85% inhibition, was chosen. A binder dilution curve for a 1:200 dilution of ADB4 was then constructed to determine an appropriate amount of avidin to use for the doseresponse studies (results not shown). Binder concentrations in the range of 0.25-0.50 pg provided sufficient inhibition of the enzymatic activity and were used to produce a family of dose-response curves (Figure 4). Typically, these curves were steep, which indicates the capability of these assays for the precise and accurate determination of biotin. The steepness of the curves is consistent with theoretial predictions of the response of enzyme immunoassays in cases where the affinity constant of the binder for the free ligand is higher than that for the conjugate (23). Optimization of these assays was undertaken by varying the concentrations of the ADA-biotin conjugate and avidin. Altering these parameters resulted in detection limits as low as 1X M biotin. As evidenced from Figure 4, when the avidin concentration is halved, the resulting curves are shifted to better detection limits by almost an entire decade. Further,
-9
-8
-7
-6
-5
log [ ~ i o t i n ] Flgure 4. Dose-response curves for biotin: (A)1:lOO dilution of ADB4 in the presence of 1 pg of avidin (static system); (0)1:200 dilution of ADB4 in the presence of 0.5 pg of avidin (automated system); (0) 1:200dilution of ADB4 in the presence of 0.25 pg of avidin (automated system); (0)1:400dilution of AD84 In the presence of 0.5 pg of avidin (automated system); (B) 1:400 dilution of ADS4 In the presence of 0.25 pg of avidin (automated system). The x axis refers to the molarity of the biotin standards. Curves were generated by plotting means of duplicate measurements.
at least for the concentrations of binder tested, essentially no effect was observed on the detection limits due to changes in the concentration of ADB4. In comparison with the automated system, the dose-response curves obtained with the static system (Figure 4) were not as steep since the concentration of the reactants (Le., conjugate and avidin) was much lower than those used in the automated system. Therefore, the latter system is more sensitive and should result in more precise assays. To quantitate the precision of the assay for biotin using the automated system, the assay WBS repeated five times with four different biotin concentrations that fall in the steep portion of the curve. The coefficient of variation (CV) for each of the biotin concentrations was calculated, and the average CV was found to be 3.1 % . In order to demonstrate the general applicability of the assay, real sample studies were performed. Biotin is an essential vitamin for both humans and animals, acting as a coenzyme in carboxylation reactions involving ATP (24). Horses, for example, need biotin to promote good hoof growth and a healthy coat. Comben et al. have demonstrated through case studies the need for biotin supplementation in a horse’s diet, particularly those with badly damaged feet. They claimed that the amount of biotin necessary to sufficiently treat unhealthy hooves is 15 mg/day (25). Horse-feed supplements containing biotin can be purchased from a number of manufacturers. By use of the assay system developed, the amount of biotin in one such feed supplement was determined. Besides biotin, this sample also contained dextrose, gelatin, dried brewer’s yeast, calcium phosphate, zinc sulfate, DL-methionine, L-lysine, pyridoxineHC1, and potassium sorbate. The estimated amount of biotin found was 19% more than that claimed by the manufacturer. Recovery studies were also performed, and the quantities of biotin used to spike the samples before extraction, 48.9 and 24.4 pg, were recovered at 100% and 88%, respectively. These recoveries imply the absence of matrix effects in these determinations. In conclusion, adenosine deaminase, an ammonia-producing enzyme, was used to develop a homogeneous competitive binding assay for biotin. Steep dose-response curves were observed. By varying the concentrationsof the assay reagents, one can adjust the steep portion of the dose-response curve to a desired value over a wide range of biotin concentrations. Finally, the approach presented in this report results in an
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Anal. Chem. 1989, 61, 1732-1736
assay that allows for potentiometric rather than photometric detection schemes and, therefore, should be advantageous for the determination of biotin in turbid and colored samples.
ACKNOWLEDGMENT We thank W. L. Gore and Associates for the generous donation of the gas-permeable membrane. Registry No. ADA, 9026-93-1; biotin, 58-85-5. LITERATURE CITED (1) Ngo, T. T.; Lenhoff. H. M. Appl. Biochem. Biotechnol. 1981, 6 , 53-64. (2) Rowley, G. L.; Rubenstein, K. E.; Huisjen, J.; Uilman, E. F. J . Biol. Chem. 1975, 250, 3759-3766. (3) Uliman, E. F.: Magglo, E. T. I n Enzyme-Immunoassay; Maggio, E. T., Ed.; CRC Press: Boca Raton, FL, 1980; pp 105-134. (4) Uliman, E. F.; Yoshlda, R. A.; Blakemore, J. J.; Maggio, E. T.; Leute, R. Biochim. Slophys. Acta 1979, 567, 66-74. (5) Heineman, W. R.; Halsali, H. 6.; Wehmeyer, K. R.; Doyle, M. J.; Wright, D. S. Methods Bbchem. Anal. 1987, 3 2 , 345-393. (6) Fonong, T.; Rechnltz, G. A. Anal. Chem. 1984. 5 6 , 2586-2590. (7) Gebauer, C. R.; Rechnltz, G. A. Anal. Bimhem. 1980, 703. 280-284. (8) Gebauer, C. R.; Rechnltz. G. A. Anal. Blochem. 1982, 724, 338-348. (9) Gebauer, C. R.; Rechnltz, G. A. Anal. Left. 1981. 74, 97-109. (10) Monroe, D. Am. Biotechnol. Lab. 1986, 4 , 28-39. (11) Fraticelli, Y. M.; Meyerhoff, M. E. Anal. Chem. 1981, 5 3 , 992-997. (12) Martin, G. B.; Meyerhoff, M. E. Anal. Chlm. Acta 1986. 786, 71-80. (13) Keeley. D. F.; Walters, F. H. Anal. Left. 1983, 16, 1581-1584.
(14) Daunert, S.;Bachas, L. G.; Meyerhoff, M. E. Anal. Chim. Acta 1988, 208, 43-52. (15) Lucacchini, A.; Bertolini, A. D.; Ronca, G.; Segnini. D.;Rossi, C. A. Biochlm. Slophys Acta 1979, 569, 220-227. (16) Brontman, S. 6. Ph.D. Thesis, University of Michigan, 1984. (17) Bachas, L. G.; University of Mlchigan, unpublished results, 1986. (18) Green, N. M.; Koniecznv, L.; Toms, E. J.; Valentine, R. C. Bbchem. J . 1971, 725,781-791. (19) Bachas, L. G.; Meyerhoff, M. E. Anal. Chem. 1988, 5 4 , 956-961. (20) Kabakoff, D. S.;Greenwood. H. M. I n Recent Advances in Clinical Biochemistry; Alberti, K. G. M., Price, C. P., Eds.; Livlngston: London, 1981: .. 00 r r 1-30 -(21) SamakB, H.; Rajkowski, K. M.; Cittanova. N. C/in. Chim. Acta 1983, 130, 129-135. (22) Zettner, A. Clin . Chem. ( Winston-Salem, N .C .) 1973, 19, 699-705. (23) Bachas, L. G.; Meyerhoff, M. E. Anal. Biochem. 1986, 756, 223-238. (24) Harris, R. S.;Gyorgy. P.; Langer, B. W. I n The Vitamins; Sebrell, W. H., Jr., Harris, R. S.,Eds.; Academic: New York, 1966; Voi. 11, pp 261-359. (25) Comben, N.; Clark, R. J.; Sutherland, D. J. 6 . Presented at the Annual Congress of The British Equine Veterinary Association, University of New York. September 5, 1983.
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RECEIVED for review December 20, 1988. Accepted April 17, 1989. This research was supported by grants from the American Association for Clinical Chemistry (Research and Endowment Fund) and from the National Institutes of Health (Grant No. R29-GM 40510).
Quantification of Recombinant Interleukin-2 in Human Serum by a Specific Immunobioassay R. W. Nadeau,* N. F. Oldfield, W. A. Garland, and D. J. Liberato Department of Drug Metabolism, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110
A sensitive and specific assay for recomblnant Interleukln-2 (rIL-2) In human serum Is descrlbed. The assay is based on a sequential sandwich Immunobloassay that uses a mlcrotlter plate coated wlth antCrIL-2 monoclonal antibody (specific for recomblnant human IL-2) to capture rIL-2 from serum, and an IL-2 dependent T-cell llne that proliferates In a dose-dependent fashlon. The lower limit of quantltation of the assay is 2 unlts/mL ( 1 unlt = -50 pg) using 0.1 mL of serum and the callbration curves ranged from 2 to 50 unlts/mL. Data are reported on the sensltivlty, preclslon, reproduclblllty, and specificity of the assay; the stabillty of rIL-2 In serum; and the recovery of rIL-2 from serum. We also report on the use of the procedure to assay clinical samples from patients with AIDS undergolng treatment with rIL-2.
INTRODUCTION Interleukin-2 (IL-2), first described as T-cell growth factor ( I ) , is a glycoprotein (15.5 kDa) of 133 amino acids with a carbohydrate moiety attached to threonine at position 3 (2). IL-2 is a lymphokine produced and secreted by T-cells when they are activated with either an antigen or mitogen (3). It plays a key immunoregulatory role by inducing proliferation and differentiation of many types of effector cells ( 4 ) . Recently, attention has focused on the clinical potential of IL-2 in cancer immunotherapy (5), and, in this regard, preclinical as well as clinical studies with IL-2 became possible only after the structural characterization and expression of a cloned gene for human IL-2 in E. coli allowed for the manufacture of unlimited amounts of the protein (6). This
manufactured IL-2 (recombinant IL-2, rIL-2), differs from the natural IL-2 in that it contains no carbohydrate functions and has an additional methionine residue a t the N-terminus. This paper describes an assay for rIL-2 in the serum of patients undergoing treatment with the protein. The assay is being used to gain the pharmacokinetic information required to establish the optimum dosing regimen for the drug and to establish a relationship between serum concentrations of rIL-2 and its efficacy and toxicity. The assay is based on the ability of rIL-2 to regulate the proliferation of IL-2 dependent mouse CTLL-2 cells (7). It uses a modification of the hybrid assay developed by Familletti et al. (8). We have used the capture antibody method because we are required to measure the concentration of teceleukin (Roche recombinant IL-2) in severely ill patients receiving various concomitant medications. The capture antibody recognizes the first 1 2 amino acids of the recombinant product as determined by Western blot and by demonstrating that pure glycosylated IL-2 does not bind to 5B1. The assay differs from similar published bioassays for IL-2 (2, 6) in that it utilizes several calibration standards to construct a calibration curve, allows for the direct addition of pure serum, utilizes a capture antibody technique to isolate rIL-2 from potentially interfering compounds, and includes a significant degree of validation.
EXPERIMENTAL SECTION Equipment. A Sterilgard hood (The Baker Co.) was used to provide aseptic conditions. Cells were maintained in a waterjacketed incubator (Forma Scientific Model 3157). Cell counts and viability were determined by using a Diaphot inverted-phase contrast microscope (Nikon). Pipeting into microtiter plates and the washing of plates was done with a Pro/Pette liquid-handling system (Perkin-Elmer). A general-purpose automatic refrigerated
0003-2700/69/0361-1732$01.50/0C 1989 American Chemical Society
