soia
Anel. Chem. 1992, 64, 3018-3023
Microtiter Plate Binding Assay for Cholinergic Compounds Utilizing the Nicotinic Acetylcholine Receptor Lu Chen, Glenn B. Martin, and Garry A. Rechnitz' Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
A receptor-based Mndlng assay for the determlnatlon of cholinerglccompound8 of the nkotlnlc acetylcholine receptor ha8 been developed. By conducting the assay In a @&well microtner plate, the methodbsuttabkfor largarcalescreening In drug development. WkLphase extractlon of the enzyme label signifkantiy dmpllfler the away protocol compared to earlier methods TRe assay lo ba8ed on ImmoMllzatlon of Motln-BSA on the mlcrotiter whkh take8 up avldln-labeled peroxidase due to avidln-blotln Interaction. To perform the assay, a llgand (the analyte) and a blotln a-bungarotoxln conjugate (aegt-blotln) sequentially Mnd to a vesicle bound nicotinic acetykhdlne receptor. Thk k done either In a te8t tube, assay I, or In a bbtlnylated mlcrotlter well, assay 11. AvldlMRP k then added to this mMure; free aBgt-blotln conJugrteand kmnobHizedWin-BSA compete for the avldln shes. After the away dution has been a8pkat.d off, bound enzyme actlvlty k detennlnedwhich k dlrectiy related to the amount of aBgt-Motln added. Dose-response curves of chollnergk compound8 and Scatchard plot8 were generated to evaluate the apparent Mndlng Cor#rtants. Klnetk 8tudh were conducted for the purpose of optknlzatlon. The Hnal assay can be performedIn under 4 h with a mlnlmm of 8ample handling.
INTRODUCTION The development of practical nonisotopic binding assays is currently an area of great analytical interest. A variety of labeling compounds,such as enzymes and fluorophores, have been utilized for the measurement of receptor-ligand interactions. More recently, enzymatic methods have been adapted to the microtiter plate reader, providing a receptor binding assay which enables hundreds of assays to be performed per day.'+ In conjunction with computer software, this type of assay is able to quickly obtain manifold results, including calculation of kinetic data. The nicotinic acetylcholine receptor (nAChFt) from electric tissue of Torpedo californica is one of the best studied neuroreceptors. It is a complex transmembrane protein, formed by homologoussubunits (a&"/& which contain agonist and antagonist binding sites (receptor function), ligand-gated cation channelsite (response function),and several other kinds of modulator sites (modulator f u n ~ t i o n ) . ~ The interaction of avidin-biotin has become very useful in a wide variety of bioanalytical applications, owing to the high (1) Baker, M. A.;Cerniglia, G. J.; Zaman,A. Anal. Biochem. 1990,190, 360-65. (2) King, I. C.; Catino, J. J. AnaZ. Biochem. 1990, 188, 97-100. (3) Cleaveland, J. S.;Kinener, P. A,; Hammond, D. J.; Schacter, B. Z. Anal. Biochem. 1990,190,249-53. (4) Fels, G.;Plumer-Wilk,R.; Schreiber,M.; Maelicke,A. J. Biol. Chem. 1986,261, 15746-54. (5) Conti-lkonooni, B. M.;Diethelm,B. M.;Wu, X.;Tang,F.;Bertezzon, T.;Schroder,B.;Reinhardt-Maelicke,S.;Maelicke, A.Biochemistry 1991, 30, 2575-84. (6) Tanaka, S.;Takeuchi, T.; Rechnitz, G . A. J. Chromatogr. 1992, 597.443-47. 0003-2700/92/0364-3018$03.00/0
affinity (1016M-'1 and stable nature of the resultingcomplex.7 The general principle of the avidin-biotin system is that biotin, coupled to another molecule or biologically active compound, can still be recognized by avidin which is in turn derivatizad with a reporter group such as a fluorophore or enzyme. As only the bicyclic ring system of the biotin molecule is required for recognition, the carboxyl group of the valeric acid side chain of biotin can be modified with assorted substances. It is now widely appreciated that the avidin-biotin system provides high sensitivity in fluorescence or enzyme-based detection, great versatility due to easy introduction of multiple markers, and a lower background signal than when proteins are labeled directly with a fluorophore. The enzyme-linked immunosorbent assay (ELISA) is a commonly employed technique in immunoaseays.8 The analyte to be detected binds either to an antigen or to an antibody coated onto a solid phase, and unbound molecules are then readily washed free from the solid support to allow measurement of the bound enzyme conjugate. Both antigen competitive inhibition, for haptens, and double antibody sandwich, for multiepitope antigens, are easily performed using the ELISA method, while antibodies can be quantitated by the indirect method. Speciallytreated polystyrene ELISA plates strongly adsorb proteins onto the wall of the wells by hydrophobic interaction, which can be utilized in immobilization of a biotin-BSA conjugate. By employing an avidin-biotin system as a label and the ELISA plate as a solid support, a receptor-based microtiter plate assay is established based on the competitive interaction between the analyte and a labeled ligand, biotinylated a-bungarotoxin, for the nAChR a-binding sites. Added avidin peroxidase then titrates the remaining free biotinylated a-bungarotoxin;any excess avidin peroxidase is extracted by the biotinylatad well. This new nonisotopic assay method can be completely carried out within a microtiter well, which simplifies the assay protocol and greatly reduces the detection time. The system proved to be simple, fast, and sensitive for the determination of cholinergic compounds of the acetylcholine receptor.
EXPERIMENTAL SECTION Apparatus. Electric organ tissue was homogenized with a Brinkmann PAT 20s (Westbury, NY). Test tubes used were FisherBrand Disposable Culture Tubes/Flint Glaee (Fisher Scientific, Pittsburgh, PA). ELISA plates were Falcon ProBind plates from Fisher Scientific. The microtiter plate reader employed was a SLT-LabInstruments Easy Reader 340AT (Austria, Europe). The shaker bath was a GCNPrecision Scientific Co. Model 25 (Chicago, IL). The spectrophotometer used was a Milton Roy Spectronic 1201 (Rochester, NY). Reagents. Electricorgan of Torpedocalifornicawas obtained from Pacific Bio-Marine Laboratory. Horseradish peroxidase avidin D (avidin-HRP, 1.5 mol of HRP/mol of avidin) was from Vector Laboratories, StabilCoat Immunoassay Stabilizer was from Bio-Metric System, Inc., and biotinylated a-bungarotoxin (7) Wilchek, M.; Bayer, E. A. AnaZ. Chem. 1988,171, 1-32. (8) Monroe, D. Anal. Chem. 1984,56,920A-931A. Ca 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
(aBgt-biotin, 1mol of biotin/mol of aBgt) was from Molecular Probes, Inc.. Hydrogen peroxide,sulfuricacid,sodiumphosphate monobasic, and NJV-dimethylformamide (DMF) were ordered from Fisher Scientific. Gallamine was purchased from Aldrich Chemical Co. All other reagents were obtained from Sigma Chemical Co. Biotinamidocaproyl-bovine serum albumin (biotin-BSA) consists of 9.6 mol of biotin/mol of BSA. Membrane bound nAChR was prepared as described in ref 9. When being used, the receptor was resuspended and diluted with PBS-BSA buffer solution (10mM sodiumphosphate, 0.1 M NaC1, 0.1 % BSA, pH 7.4). The aBgt-biotin (0.5 mg) was reconstituted in 0.5mL of deionized water to make a 1.2 X lo4 M stocksolution, and aliquots were stored at -20 "C. Solid biotin-BSA (1mg) was dissolved in 100 mL of PBS to obtain a concentration of 0.001% (w/w). Avidin concentration from commercial avidin-HRP is 5 mg/mL, which is approximately equal to 4 X M. There are two kinds of buffer involved during the assay, one is PBS-BSA which was used to prepare all assay samples, the other one is PBS (10 mM sodium phosphate, 0.1 M NaCl, PH 7.4) which was for washing of the plates. In the enzyme activity determination, the TMB substrate solution was prepared by dissolving 10 mg of 3,3',5,5'-tetramethylbenzidine in 1 mL of DMF and 100 mL of 0.1 M sodium acetate (pH 5.5). Procedures. Immobilization ofBiotin-BSA to the Microtiter Plate. Immobilization of biotin-BSA to the microtiter plate was achieved by adsorption. First, 100 pL of biotin-BSA (0.001% ) was pipetted into the wells and incubated overnight at 4 "C. The plates were then rinsed with PBS three times after the biotinBSA had been aspirated off. Two different substances were employedto block the possible nonspecific binding onto the walls, i.e., 1%BSA in PBS solution (for assay I) or StabilCoat (for assay 11). In both cases, 250 pL was added to the plates and incubated for 45 min at room temperature. The biotin plates prepared by this method can be stored for at least 1week in a dry state at 4 "C without significant loss of activity. Solid-Phase Competitive Binding Assay I (Assay I). For reasons previously discussed, two parallel sets of experiments must be performed to obtain a saturation curve.g The first set of samples was run to determine the total binding (specific and nonspecific) of aBgt-biotin to the receptor. A 50-pL volume of 20-fold diluted acetylcholine receptor preparation was incubated with 400 pL of various concentrations of aBgt-biotin, which were made up in PBS-BSA buffer, in test tubes for 2 hat 4 "C. AvidinHRP (50 pL) was then added to produce a final concentration of 0.2 nM. This mixture was incubated at 4 "C with shaking for 1 h. A 100-pL aliquot of this solution was pipetted into a biotinylated well and followed by another hour of incubation (Figure 1). The secondset of sampleswas run to determine the nonspecific binding. The nAChR (50 pL) was first allowed to bind with M) for 30 carbamylcholine (50pL, final concentration 5 X min and then incubated with the various concentrations of aBgtbiotin for another 2 h. All the other steps were kept the same as for the first set. Carbamylcholine functions as a specific displacer occupying all the a-binding sites of the nAChR during the incubation; consequentlyaBgt-biotin can only nonspecifically bind to the nAChR preparation. For displacement studies, 50 pL of several different concentrations of each analyte were incubated with the nAChR in test tubes for 2 h, followedby addition of aBgt-biotin and incubation of the mixture for another hour with constant shaking at 4 "C; these were found to be the optimal conditionsin previous work.9 The rest of the procedure, includingthe additionof 0.2 nM avidinHRP and aliquot transfer to the microtiter plate, was carried out as described above. Solid-Phase Competitive Binding Assay 11 (Assay II). The principle of assay I1 is the same as the assay I, except that all incubation steps wereperformed in the biotinylatedplate directly instead of in test tubes. Again, the first set of the samples was run to determine the total binding (specific and nonspecific). First, 50 pL of diluted nAChR solution and 50 pL of various concentrations of aBgt-biotin were pipetted into the wells. PBSBSA was added to bring the total volume to 150 pL. After 2 h of incubation at 4 "C with shaking, 50 pL of avidin-HRP (9) Chen, L.; Takeuchi, T.; Rechnitz, G. A. Anal. Chim. Acta, in press.
nAChR
e
3019
nAChFt aBgt-biotin
(1)
r
1 E E E
AAA, avidin-HRP (21
wash (4)
biotinylated well (3)
v
r-i
enzyme activity determination
r
(5)
Figure 1. A schematic diagram of the microtiter plate binding assay principle: (1) receptor a-sites bind with labeled ligand aBgt-biotin; (2) added avidin-HRP titratesthe remainingfree am-biotin; (3) biotinylated well extractsthe excess added avidin-HRP; (4) assay solution is washed off; (5) substrate is added to bound enzyme.
was added (finalconcentration 0.2 nM) and incubated for 1more hour. In the second set of samples for determination of the nonspecific binding, 50 pL of receptor was first allowed to bind with 50 p L of carbamylcholine (final concentration 5 X lW3 M) for 30 min and then incubated with 50 pL of aBgt-biotin for 2 h. Avidin-HRP was finally added into the sample followed by another hour of incubation. Because avidin-HRP binding to biotin-BSA on the solid support is much slower than that to aBgt-biotin in the solution,1°these two interactions are roughly noncompetitive. Displacement experiments were conducted in the same way except that the nAChR was first incubated with 50 pL of the analyte in place of carbamylcholine, and the incubation time was extended by 30 min to 1 h. Enzyme Activity Determination. After sucking all of the solution out of the wells, the ELISA plate was washed five times with PBS. The enzyme reaction was then started by adding 100 pL of TMB and 50 pL of H202 (0.01%) into the well. The substrate solution was incubated at room temperature with shaking. After 20 min, 50 pL of 0.2 N H2SO4 was added to stop the reaction, and the absorbance at 492 nm was measured with a microtiterplate reader? This detectionwavelengthwas chosen because it yielded the largest change in absorbance among the filter wavelengths available on the plate reader. Unless stated otherwise, all assay sampleswere run in triplicate, and the results were averaged to obtain the reported value. The enzyme activity varies somewhat from day to day even with the same amount of avidin-HRP bound onto the wall. Therefore, to standardize the data, results are expressed as % (percentage) enzyme activity. The value for 100% enzyme activity in each experiment was obtained by incubating 0.2 nM avidin-HRP PBS-BSA solution in the biotinylated microtiter plate under the same experimental conditions.
RESULTS AND DISCUSSION Biotinylation of ELISA Wells and Its Binding with Avidin-HRP. A standard protocol was employed to coat the plate without further optimization. Alarge excess of BSA (0.001%) was used to speed up the coating process. By controlling the incubation time, only a fixed amount of biotinBSA is immobilized on each well, which can be quantified (10) Takeuchi, T.; Rechnitz, G. A. Bioconj. Chem. 1990,1/4,227-230.
9020
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 14
12 10
8 6
1
0
2
I
4
-1 aBgt-blotln] ( n M
4
2
0’ 1 2 6 8 10 12
0.0
4
0
-’
avldln-HRP ( x i 0
0
mol)
Figure 2. Blndlng of avMin-HRP to the ELISA plate. The MTP was coated wlth 0.001 % biotin-BSA by incubating overnlght, blocked with 1% BSA for 45 mln at room temperature, and Incubated wlth avMlnHRP for 1 h at 4 O C wlth shaking. 0.6
I
1
0.4 0.3 -40
-
0.1
0.0
A
0
I
I
I
I
I
1
2
3
4
5
I
I
I
I
1
4
6
8
10
12
ll[aBgt-biotin] ( n M - ’ ) Figure 4. Enzyme acthrity (%) vs [aBgt-biotin] curve for the determination of the amount of “bound”and “free”labeled Ilgands. Small (inset): obtained by fixing [avidin-HRP] at 0.2 nM while varylng
the amount of aBgt-biotin present In the solution. Large: reciprocal plot, relative acthrlty = enzyme actlvlty/maxlmum enzyme actlvlty (obtained from the samples when only 0.2 nM avidin-HRP was introduced) = (% enzyme acthrlty)/100. (0)Assay I, y = 0.944 0 . 7 8 4 ~(X) Assay 11, y = 0.947 0.243~.
+
0.5
0.2
I
2
20
a2
d
10
6
incubation time @) Figure 8. The effect of incubation time on uptake of avidin-HRP onto the blotinylated plate at 4 O C . [avMin-HRP] = 0.2 nM (2 X lo-” mol).
Percent (%) enzyme actlvlty = enzyme acthrlty/maximum enzyme acthrky (obtained from a 5-h Incubation).
by avidin-HRP binding. Excess BSA is then removed by aspirating the coating solution. Protein concentrations higher than 0.001% should be avoided so that excessive leaching does not occur.11 The biotin-binding activity of avidin-HRP and the doseresponse curve for inhibition of its binding to biotin-BSA by free biotin have been previously reported.10 The binding behavior of avidin-HRP with biotinylated wells was studied by incubating various amountsof avidin-HRP in a microtiter plate. As shown in Figure 2, the binding exhibits a saturable behavior for avidin-HRP above 6 X 10-14mol. The absorbance was limited to about 0.6 because of the amount of biotinBSA immobilized and the incubation time with substrate solution. In the range from 0 to 2 X 10-14mol, the quantity of avidin-biotin bound onto the solid support is proportional to the moles of avidin-HRP present in the solution. Therefore, 2 X lO-l4 mol, which is 0.2 nM when converted to concentration, was chosen for later studies as it afforded the greatest sensitivity. The incubation time dependence of avidin-HRP binding to the solid support is demonstrated in Figure 3. The amount of binding reached a maximum only after 5 h; however, at 1 h approximately 80% of maximum was achieved. The relatively long incubation time is required for the binding of avidin to biotinylated wells because of the heterogeneous nature of the interaction. Determination of the Bound and Free Labeled Ligands. In order to obtain the amount of “free”and “bound” (11) Campbell, A. M. Laboratory Techniques in Biochemistry and Molecular Biology-Monoclonal A n t i b o d y Technology; Elsevier Biomedical: Amsterdam, 1983; Volume 13.
+
labeled ligands, a percent enzyme activity vs [aBgt-biotin] curve was generated by fixing the avidin-HRP concentration at 0.2 nM while varying the amount of aBgt-biotin in the solution (Figure 4, inset). Thus as long as one knows the percent enzyme activity, the correspondingfree aBgt-biotin concentration in the assay sample can be determined from this curve. The results from the first set of experiments (see the ExperimentalSection)provide the “free”signals and thoee from the second set provide the “total”signals. The difference between the “free” and “total” signals is the “bound” signal. For the purpose of accurately calculating the aBgt-biotin concentration from a given response, the raw responses from the inset of Figure 4were linearized by means of two reciprocal plots (Figure 4, large). These plots have a form similar to those of Lineweaver-Burk.12 The difference between the two curves in Figure 4 is an interesting phenomena. The possible source of this difference is mainly due to the change in the experimental design when going from assay I to assay 11. In the first assay, avidin-HRP and aBgt-biotin binding reaches an equilibrium before an aliquot of the solution is added to the microtiter plate and incubated for 1 h. However, in the second case both the avidin-HRP/aBgt-biotin and avidin-HRP/immobilized biotin binding occurs simultaneously in the microtiter plate, with a total incubation time of just 1h. Thus the binding between avidin-HRP and aBgt-biotin may not reach a true equilibrium due to the presence of immobilized biotin on the microtiter plate, and consequentlythere will be more avidinHRP bound to the walls. As a result, the amount of avidinHRP bound to the microtiter plate is higher in assay I1 than in assay I. However, this difference does not in any way appear to compromise the results of either assay. Solid-Phase Competitive Binding Assay I (Assay I). Effectof Carbamylcholine Concentration. From previously published work? carbamylcholine was shown to be useful as a specific displacer to displace specific binding of the labeled ligand so that the labeled ligand could only bind nonspecifically with the receptor. Thus the carbamylcholine concentration must be high enough to displace all the specificbinding but not so high as to displace nonspecifically bound aBgt(12)Guilbault, G. G. Analytical UsesofImmobilized Enzymes,Marcel Dekker, Inc.: New York, 1984; Chapter 1. (13)Blanchard, S.G.; Quast, U.; Reed, K.; Lee, T.; Schimerlik, M. I.; Vandlen, R.; Claudio, T.; Strader, C. D.; Moore, H. H.; Rafbry, M. A. Biochemistry 1979, 18, 1875-83. (14)Taylor, P.;Brown, R. D.; Johnson, D. A. Cum. Top. Membr. Trawp. 1983, 18, 407-43.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
3021
Table I. Effect of Carbamylcholine Concentration on the Enzyme Activity % enzvme activitv ~~
[carbamylcholine] $4)
assay I
assay I1
0 0.0005
100
100
0.006
98
48
0.05
41
79
Table 11. Interaction between nAChR and Avidin-HRP % enzyme activity lnAChRl (nM) wlcentrifwation w/o centrifugation 0.2 0.4 0.8
93
90
101 100
87
98
biotin; this concentration range was demonstrated to be 0.1 M. In this system, we found that when the to 1 X carbamylcholine concentration was above 5 X M, the enzyme activity decreased significantly (Table I). The experiment was conducted by incubating avidin-HRP (0.8 nM) with carbamylcholine for 2.5 h in teet tubes and then transferring it into the microtiter plate for another hour. Therefore, a carbamylcholine concentration of 5 X 10” M was chosen in assay I for displacement of uBgt-biotin from the specific binding sites. The possible reasons responsible for the observed decrease in enzyme activity are that carbamylcholine inhibits HRP activity,hinders avididbiotin binding, or displacesthe biotinBSA coated on the solid support. The fvst possibility was investigated by incubating avidin-HRP with and without carbamylcholine in cuvettes for 2 h and then determining the absorbance through a spectrophotometer. The results indicate that carbamylcholine does not affect HRP activity. As a matter of fact, the observed phenomena is mainly due to the displacement of biotin-BSA from the walls of the microtiter plate, which is proved in later studies. Consideration of Possible Nonspecific Interactions. Nonspecificinteraction of both the nAChR and avidin-HRP with the biotinylated plates proved to be negligible with an incubation time of 4 h. The avidin-HRP activity, however, could be affected by the presence of nAChR preparation, which was studied by two parallel experiments. In the fist set of experiments the supernatant of the nAChR preparation was centrifuged (27000g, 10 min) and the supernatant was used, while in the second case the crude nAChR preparation was employed directly without centrifugation. These two samples were incubated with avidin-HRP in test tubes for 1h and then in a microtiter plate for another hour. In Table 11,the enzyme activity shows decreasing trends in both cases, which is due to the nonspecificinteraction between the nAChR preparation and avidin-HRP so that less free avidin-HRP is available to bind with the plate. The result suggests that avidin-HRP nonspecifically interacta not onlywith the nAChR membrane, but also with the biological matrix present in the supernatant after centrifugation. However, the magnitude of this interaction is not significant even for the concentration of 0.8 nM used in all other studies if no centrifugation is employed. On the other hand, the enzyme activity difference between the samples with and without centrifugation is significant. The lower enzyme activity bound to the microtiter plate when the receptor preparation is centrifugedis likely due to adsorption of avidin-HRP to the walls of the test tube, which was used to incubate the avidin-HRP with the supernatant, before pipetting an aliquot into the microtiter plate. Avidin-HRP adsorption does not occur in the test tube if the receptor
40
’
0
I
I
20
40
I 60
I
80
I
I
I
I
100 120 140 160
incubatlon time (mln) F l g w 1. Optlmlzetkn of incubatlon time of aWn-HRP/free aBgtbktin bkrdkrg after the receptor ha8 been equilibrated wtth carbam ylcholine~and uBgt-blotin. [nAChR] 0.8 nM, [carbamylchollne] = 5 X 109 M, [aBgt-blotln] = 0.5 nM. (X) nAChR aBgt-bktin; (0) nAChR carbamyichoiine aBgt-blotln. Selected incubatkn time 60 min.
+
+
+
preparation is not centrifuged, owing to the presence of a high concentrationof proteins and surfactants in the receptor matrix. Optimization of the Incubation Times. Figure 5 shows the effect of incubation time on the avidin-HRP/aBgt-biotin binding after the receptor has been equilibrated with either uBgt-biotin or both carbamylcholine and uBgt-biotin. The percent enzyme activity difference obtained from two sets of experiments has almost reached a maximum after 60 min; this was fued as the incubation time for later experiments. The question of whether a significant amount of binding occurs between avidin-HRP and nAChR/uBgt-biotin complex was addressed by a kinetic study. An excess amount of nAChR (0.8 nM) was mixed with uBgt-biotin (0.1 nM), and then avidin-HRP was added to the mixture and incubated for different periods of time. The result shows that at up to 2-h incubation, enzyme activity is only decreased about 5% ; thus, the rate of formation for the nAChRIuBgt-biotin/ avidin-HRP complex is negligible. This is desirable because it demonstrates that avidin-HRP effectivelyonly binds with free aBgt-biotin in the aqueous phase and the biotin-BSA on the solid support. Therefore a step to separate free from bound uBgt-biotin is not needed to perform this w a y . Once the assay sample has been pipetted into a biotinylated well, Figure 3 illustrates that the maximum binding between the avidin-HRP and the solid support occurs after 5 h. However, because of the fact that the longer the incubation time, the more dissociation of the aBgt-biotin/ avidin-HRP complex and association of avidin-HRP with nAChR/uBgt-biotin, a 1-h incubation time was chosen for this step. Saturation Curve and Scatchard Plot. Figure 6 depicta typical curves obtained from assay I when varying the concentration of aBgt-biotin. The enzymeactivitydifference between sets 1 and 2 is due to the specific binding of the nAChR and uBgt-biotin, while that between sets 2 and 3 is due to the nonspecific binding. Using the relationship shown in Figure 4,all the percent enzyme activitywas converted into the correspondingamount of “free” and ‘bound” labeled ligand. Thus the saturation curve and Scatchard plot could be generated (Figure 7). From the Scatchard plot, the apparent dissociation constant WBB determined to be 9.4 X 10-11 M, and the maximum uBgtbiotin binding sites was 19nmol/g protein or 0.77 nM of aBgtbiotin binding sites. Comparing these values to those from a previous study? the & is around 1 order of magnitude lower, which may partly arise from the binding affiiity difference between uBgt-biotin, used in this work, and uBgt-
3022
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23,DECEMBER 1, 1992 100 80
U
a
m a9
60
40 20
0
1
2
3
4
5
6
-11.10-9 - 8 - 7
[~Bgt-biotinl[nMl Figure 6. compewhrebinding assay I cvv88obtahedby fixing [nAchR] at 0.8 nM, [carbamylcholine] at 5 X lo3 M, [avidin-HRP] at 0.2 nM, and varying the concentratbn of aBgt-biotin: (A)set 1, nAChR aBgt-biotin; (0) set 2,nAChR + cerbamylchollne aBgt-blotln; (X) set 3, aBgt-biotin only.
+
+
250 200 150 100
50
0
I
I
I
5
10
15
20
Bound (nmoilg protein)
Flguro7. Scatchard analysis for assay I. Same conditions as Flgure 6. Small (Inset): Saturation curve. Large: Scatchard plot. From the plot, & = 9.4 X lo-“ M, and S,= 19 nmol/g of protein = 0.77 nM of aBgt-biotin sites.
FITC, used in the previous work, with the receptor. The apparent dissociation constant of aBgt-biotin compares very favorably with the range of values reported in the literature (10”J-10-12)for aBgt itself, which indicates that the activity of aBgt-biotin is very similar to that of aBgt. Dose-Response Curves. Doseresponse curves for aBgt and cholinergic ligands were obtained (Figure 8) by using a labeled ligand concentration of 0.5 nM which results in the maximum percent enzyme activity difference between experimental sets 1and 2 (see Figure 6). Apparent dissociation constants can be subsequentlycalculated using the ICs0 values obtained from the displacement curves. These data are consistent with literature values using radiolabeled receptorbased assays and show lower detection limit than the fluorophore-labeled nAChR assay (Table 111). The reproducibility of the dose-response curves proved to be satisfactory, with no more than 20% error at the 90% confidence interval (N= 6 ) . The main sources of error are likely to be from the irreproducibility of the biotin-BSA coating from well to well and the accumulation of deviation caused by the multiple incubation steps involved. Solid-Phase Competitive Binding Assay (Assay 11). Effect of Carbamylcholine Concentration. The most difficult problem encountered in the development of the assay I1 method was the significant reduction of the enzyme activity bound to the plate even if a carbamylcholineconcentration as low as 5 X lo4 M was used (Table I). This is well below the concentrationof carbamylcholine needed to be an effective specific displacer. From the discussions above, we were
-6 - 5 - 4 -3 - 2 --1
0
1
2
log[anaiyte] (M) Figure 8. Dose-responsecurves of agonists and antagoniets to the a-subunttsof the nAChR (assay I). [nAChR] = 0.8 nM, (aBgt-bktln] = 0.5 nM, and [avidin-HRP] = 0.2 nM. Percent (%) bound (specmC binding) = bound/mdmum bound (obtained form the assay eempks when no dieplacers were Introduced): (A)aBgt;(0)pancuronlum; (+) ~~(~)Slalamine;(X)cerbemyldrdlne;(O)decemahonknn; ( 0 )bxamethonium. Small (inset): reproducibility of cerbamylcholhre curve. Error bare (90% confidence interval)were obtained for 6 Mais.
certain that carbamylcholine did not obstruct the activity of HRP. It was suspected that this effect was most probably caused by the displacementof the biotin-BSA adsorbed onto the solid support, and the magnitude of this displacement would increase with the incubation time. In w a y 11, carbamylcholine was allowed to stay in contact with the microtiter plate for 3.5 h instead of only 1h as was the case in assay I, which explains why the displacement was greatly increased in assay I1 even though the carbamylcholine concentration was 5 X 10-3 M in both cases. In order to solve this problem, the StabilCoat immunoassay stabilizer was utilized as asubatitute for the 1% BSA solution in the blocking step of the biotinylation procedure.16 The use of StabilCoat returned the amount avidin-HRP activity to 98% for the 3.5-h incubation; this is similar to the values obtained with the 1h incubation time used in assay I. This experiment also eliminated the possibility that carbamylcholiie hinders avidin-biotin binding. The StabilCoat ingredient interads with active biomolecules immobilized or adsorbed onto a surface and maintains their biological activity under extreme conditions such as high surfactant levels and high temperatures. It can be used to replace the blocking step in immunoassays, and coated ELISA plates retain nearly 100% activity for long as 5 months at 40 “C. Scatchord Anulysis. Under assay I1 experimental conditions, Figure 9 depicts the typical curves obtained when a f i e d amount of receptor is incubated with various concentrations of aBgt-biotin. The amount of ‘bound” and “free* were determinedfrom Figure 4 according to the correaponding percent enzyme activity, and from these data both the saturation curve and the Scatchard analysis were constructed (Figure 10). The & value obtained from the Scatchard plot is 9.6 X l@ll M which is almost the same as that from assay I. B , is 23 nmoVg of protein, Le., 0.91 nM of arBgt-biotin binding sites, which is also close to the result from assay I and well within the range of experimental deviation (the average concentration from assays I and I1 is 0.84 nM aBgt-biotin sites). However, because the assay was conducted directly in amicrotiter plate, the procedureis easier and more convenient for the purpose of automation; as an added bonus the time needed to perform the assay is shortened by 1h. Dose-Response Curves. Figure 11gives the dose-response curves of aBgt and various cholinergic ligands obtained using (15)StabilCoat. Immunoassay Stabilizer Product Information; BioMetric Systems, Inc.: Eden Prairie, MN.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
8028
Table 111. Apparent Dissociation Constants of Agonists and Antagonists Obtained from Assays I and I1 RRA (lit.) assay I
anal* ffBgt
7.4 x 6.6 X 5.3 x 3.3 x 2.6 X 8.4 X 2.1 x
carbamylcholine d-tubocurarine pancuronium gallamine decamethonium hexamethonium
assay I1 9.4 x 6.7 X 4.7 x 3.3 x 3.0 X 8.4 X 1.9 x
10-10
lW7 10-7
10-8 10-8 10-1 10-4
ref 13
ref 14
10-10
lo1 lo-'
1.2 x 10-7 2.0 x 10-7
10-8
10-6 10-1 lo-'
1.8 X 10-1 1.2 x 10-4
I
0
1
2
4
3
5
6
I W t - b i o W (nM) Flgure 8. Competittve binding assay I1 curves, obtained under the same conditions as Figure 6; (A)set 1, nAChR aBgt-biotin; (e)set 2. nAChR carbamyichoiine aBgt-biotin; (X) set 3, aBgt-biotin
only.
+
+
+
300
250 200 150 100
50 0
0
5
10
15
20
25
B o u n d (nmoi/g protein) Flgure 10. Scatchard analysis for assay 11. Small (inset): saturation curve. Large: Scatchard plot. From the plot, & = 9.6 X lo-" M, and S,= 24 nmol/g of protein = 0.91 nM of a-subunit.
assay 11. The apparent dissociation constants are calculated from the IC@ obtained from these curves (Table 111) and are almost identical with those calculated using the data from assay I. The deviation associated with the method is relatively large compared with that from assay I, showing a maximum error of 30% at 90% confidence interval (N= 6). The procedures of the two assay methods discussed in this paper are less complicated in both procedure and instrumentation compared to the previously reported FRA.g With
4.8 x 3.8 X 2.3 X 1.1x 8.3 X
10-7
10-1 10-8 10-6 10-1
FRA (ref 9) 9XlO-B 3Xlod 8XlO-B 3 X 1W7 4XlO-B BXlod 2x104
100 7
- 1 1 1 0 - 9 - 8 - 7- 6 - 5 - 4 - 3 - 2 - 1 0 1
2
log[analyte] (M) Flgurr 11. Dose-response curves of agonists and antagoniststo the a-subunits of the nAChR (assay 11).same conditions given for Flgwe 8: (A)aBgt;(0)pancwonium; (+) &tubocurarine; (e) gallamine; (X) carbamylcholine; (e)decamethonium; (0)hexamethonium. Small (inset): reproducibility of carbamylcholine curve. Error bars (90% confidence interval) were obtained for slx trlals.
the relatively short incubation time and the semiautomated capabilities of microtiter plate technology, screening of large numbers of cholinergic compounds can be easily conducted. The utilization of a solid phase for the complete separation of labeled ligand from the biological matrix simplifies the assay protocol and reduces the background signal, thereby enhancingthe sensitivity. The methodology is new and quite practical for the determination of agonists and antagonista of the nicotinic acetylcholine receptor. Moreover, use of avididbiotin labeling system should be easily adaptable to other receptor assays. To further simplify the assay protocol, solubilized acetylcholine receptors are expected to be directly immobilized onto the microtiter plate either by hydrophobic interactions or by covalent bonding. Alternatively, if a fluorescence microtiter plate reader, instead of a W/vis plate reader, is employed, fluorescence labels can replace the avidinbiotin system so that the assay format will become even less complicated.
ACKNOWLEDGMENT We gratefully acknowledge the support of NIH Grant GM25308. RECEIVED for review May 19, 1992. Accepted September 21, 1992.
Anel. Chem. 1992, 64, 3018-3023
Microtiter Plate Binding Assay for Cholinergic Compounds Utilizing the Nicotinic Acetylcholine Receptor Lu Chen, Glenn B. Martin, and Garry A. Rechnitz' Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
A receptor-based Mndlng assay for the determlnatlon of cholinerglccompound8 of the nkotlnlc acetylcholine receptor ha8 been developed. By conducting the assay In a @&well microtner plate, the methodbsuttabkfor largarcalescreening In drug development. WkLphase extractlon of the enzyme label signifkantiy dmpllfler the away protocol compared to earlier methods TRe assay lo ba8ed on ImmoMllzatlon of Motln-BSA on the mlcrotiter whkh take8 up avldln-labeled peroxidase due to avidln-blotln Interaction. To perform the assay, a llgand (the analyte) and a blotln a-bungarotoxln conjugate (aegt-blotln) sequentially Mnd to a vesicle bound nicotinic acetykhdlne receptor. Thk k done either In a te8t tube, assay I, or In a bbtlnylated mlcrotlter well, assay 11. AvldlMRP k then added to this mMure; free aBgt-blotln conJugrteand kmnobHizedWin-BSA compete for the avldln shes. After the away dution has been a8pkat.d off, bound enzyme actlvlty k detennlnedwhich k dlrectiy related to the amount of aBgt-Motln added. Dose-response curves of chollnergk compound8 and Scatchard plot8 were generated to evaluate the apparent Mndlng Cor#rtants. Klnetk 8tudh were conducted for the purpose of optknlzatlon. The Hnal assay can be performedIn under 4 h with a mlnlmm of 8ample handling.
INTRODUCTION The development of practical nonisotopic binding assays is currently an area of great analytical interest. A variety of labeling compounds,such as enzymes and fluorophores, have been utilized for the measurement of receptor-ligand interactions. More recently, enzymatic methods have been adapted to the microtiter plate reader, providing a receptor binding assay which enables hundreds of assays to be performed per day.'+ In conjunction with computer software, this type of assay is able to quickly obtain manifold results, including calculation of kinetic data. The nicotinic acetylcholine receptor (nAChFt) from electric tissue of Torpedo californica is one of the best studied neuroreceptors. It is a complex transmembrane protein, formed by homologoussubunits (a&"/& which contain agonist and antagonist binding sites (receptor function), ligand-gated cation channelsite (response function),and several other kinds of modulator sites (modulator f u n ~ t i o n ) . ~ The interaction of avidin-biotin has become very useful in a wide variety of bioanalytical applications, owing to the high (1) Baker, M. A.;Cerniglia, G. J.; Zaman,A. Anal. Biochem. 1990,190, 360-65. (2) King, I. C.; Catino, J. J. AnaZ. Biochem. 1990, 188, 97-100. (3) Cleaveland, J. S.;Kinener, P. A,; Hammond, D. J.; Schacter, B. Z. Anal. Biochem. 1990,190,249-53. (4) Fels, G.;Plumer-Wilk,R.; Schreiber,M.; Maelicke,A. J. Biol. Chem. 1986,261, 15746-54. (5) Conti-lkonooni, B. M.;Diethelm,B. M.;Wu, X.;Tang,F.;Bertezzon, T.;Schroder,B.;Reinhardt-Maelicke,S.;Maelicke, A.Biochemistry 1991, 30, 2575-84. (6) Tanaka, S.;Takeuchi, T.; Rechnitz, G . A. J. Chromatogr. 1992, 597.443-47. 0003-2700/92/0364-3018$03.00/0
affinity (1016M-'1 and stable nature of the resultingcomplex.7 The general principle of the avidin-biotin system is that biotin, coupled to another molecule or biologically active compound, can still be recognized by avidin which is in turn derivatizad with a reporter group such as a fluorophore or enzyme. As only the bicyclic ring system of the biotin molecule is required for recognition, the carboxyl group of the valeric acid side chain of biotin can be modified with assorted substances. It is now widely appreciated that the avidin-biotin system provides high sensitivity in fluorescence or enzyme-based detection, great versatility due to easy introduction of multiple markers, and a lower background signal than when proteins are labeled directly with a fluorophore. The enzyme-linked immunosorbent assay (ELISA) is a commonly employed technique in immunoaseays.8 The analyte to be detected binds either to an antigen or to an antibody coated onto a solid phase, and unbound molecules are then readily washed free from the solid support to allow measurement of the bound enzyme conjugate. Both antigen competitive inhibition, for haptens, and double antibody sandwich, for multiepitope antigens, are easily performed using the ELISA method, while antibodies can be quantitated by the indirect method. Speciallytreated polystyrene ELISA plates strongly adsorb proteins onto the wall of the wells by hydrophobic interaction, which can be utilized in immobilization of a biotin-BSA conjugate. By employing an avidin-biotin system as a label and the ELISA plate as a solid support, a receptor-based microtiter plate assay is established based on the competitive interaction between the analyte and a labeled ligand, biotinylated a-bungarotoxin, for the nAChR a-binding sites. Added avidin peroxidase then titrates the remaining free biotinylated a-bungarotoxin;any excess avidin peroxidase is extracted by the biotinylatad well. This new nonisotopic assay method can be completely carried out within a microtiter well, which simplifies the assay protocol and greatly reduces the detection time. The system proved to be simple, fast, and sensitive for the determination of cholinergic compounds of the acetylcholine receptor.
EXPERIMENTAL SECTION Apparatus. Electric organ tissue was homogenized with a Brinkmann PAT 20s (Westbury, NY). Test tubes used were FisherBrand Disposable Culture Tubes/Flint Glaee (Fisher Scientific, Pittsburgh, PA). ELISA plates were Falcon ProBind plates from Fisher Scientific. The microtiter plate reader employed was a SLT-LabInstruments Easy Reader 340AT (Austria, Europe). The shaker bath was a GCNPrecision Scientific Co. Model 25 (Chicago, IL). The spectrophotometer used was a Milton Roy Spectronic 1201 (Rochester, NY). Reagents. Electricorgan of Torpedocalifornicawas obtained from Pacific Bio-Marine Laboratory. Horseradish peroxidase avidin D (avidin-HRP, 1.5 mol of HRP/mol of avidin) was from Vector Laboratories, StabilCoat Immunoassay Stabilizer was from Bio-Metric System, Inc., and biotinylated a-bungarotoxin (7) Wilchek, M.; Bayer, E. A. AnaZ. Chem. 1988,171, 1-32. (8) Monroe, D. Anal. Chem. 1984,56,920A-931A. Ca 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
(aBgt-biotin, 1mol of biotin/mol of aBgt) was from Molecular Probes, Inc.. Hydrogen peroxide,sulfuricacid,sodiumphosphate monobasic, and NJV-dimethylformamide (DMF) were ordered from Fisher Scientific. Gallamine was purchased from Aldrich Chemical Co. All other reagents were obtained from Sigma Chemical Co. Biotinamidocaproyl-bovine serum albumin (biotin-BSA) consists of 9.6 mol of biotin/mol of BSA. Membrane bound nAChR was prepared as described in ref 9. When being used, the receptor was resuspended and diluted with PBS-BSA buffer solution (10mM sodiumphosphate, 0.1 M NaC1, 0.1 % BSA, pH 7.4). The aBgt-biotin (0.5 mg) was reconstituted in 0.5mL of deionized water to make a 1.2 X lo4 M stocksolution, and aliquots were stored at -20 "C. Solid biotin-BSA (1mg) was dissolved in 100 mL of PBS to obtain a concentration of 0.001% (w/w). Avidin concentration from commercial avidin-HRP is 5 mg/mL, which is approximately equal to 4 X M. There are two kinds of buffer involved during the assay, one is PBS-BSA which was used to prepare all assay samples, the other one is PBS (10 mM sodium phosphate, 0.1 M NaCl, PH 7.4) which was for washing of the plates. In the enzyme activity determination, the TMB substrate solution was prepared by dissolving 10 mg of 3,3',5,5'-tetramethylbenzidine in 1 mL of DMF and 100 mL of 0.1 M sodium acetate (pH 5.5). Procedures. Immobilization ofBiotin-BSA to the Microtiter Plate. Immobilization of biotin-BSA to the microtiter plate was achieved by adsorption. First, 100 pL of biotin-BSA (0.001% ) was pipetted into the wells and incubated overnight at 4 "C. The plates were then rinsed with PBS three times after the biotinBSA had been aspirated off. Two different substances were employedto block the possible nonspecific binding onto the walls, i.e., 1%BSA in PBS solution (for assay I) or StabilCoat (for assay 11). In both cases, 250 pL was added to the plates and incubated for 45 min at room temperature. The biotin plates prepared by this method can be stored for at least 1week in a dry state at 4 "C without significant loss of activity. Solid-Phase Competitive Binding Assay I (Assay I). For reasons previously discussed, two parallel sets of experiments must be performed to obtain a saturation curve.g The first set of samples was run to determine the total binding (specific and nonspecific) of aBgt-biotin to the receptor. A 50-pL volume of 20-fold diluted acetylcholine receptor preparation was incubated with 400 pL of various concentrations of aBgt-biotin, which were made up in PBS-BSA buffer, in test tubes for 2 hat 4 "C. AvidinHRP (50 pL) was then added to produce a final concentration of 0.2 nM. This mixture was incubated at 4 "C with shaking for 1 h. A 100-pL aliquot of this solution was pipetted into a biotinylated well and followed by another hour of incubation (Figure 1). The secondset of sampleswas run to determine the nonspecific binding. The nAChR (50 pL) was first allowed to bind with M) for 30 carbamylcholine (50pL, final concentration 5 X min and then incubated with the various concentrations of aBgtbiotin for another 2 h. All the other steps were kept the same as for the first set. Carbamylcholine functions as a specific displacer occupying all the a-binding sites of the nAChR during the incubation; consequentlyaBgt-biotin can only nonspecifically bind to the nAChR preparation. For displacement studies, 50 pL of several different concentrations of each analyte were incubated with the nAChR in test tubes for 2 h, followedby addition of aBgt-biotin and incubation of the mixture for another hour with constant shaking at 4 "C; these were found to be the optimal conditionsin previous work.9 The rest of the procedure, includingthe additionof 0.2 nM avidinHRP and aliquot transfer to the microtiter plate, was carried out as described above. Solid-Phase Competitive Binding Assay 11 (Assay II). The principle of assay I1 is the same as the assay I, except that all incubation steps wereperformed in the biotinylatedplate directly instead of in test tubes. Again, the first set of the samples was run to determine the total binding (specific and nonspecific). First, 50 pL of diluted nAChR solution and 50 pL of various concentrations of aBgt-biotin were pipetted into the wells. PBSBSA was added to bring the total volume to 150 pL. After 2 h of incubation at 4 "C with shaking, 50 pL of avidin-HRP (9) Chen, L.; Takeuchi, T.; Rechnitz, G. A. Anal. Chim. Acta, in press.
nAChR
e
3019
nAChFt aBgt-biotin
(1)
r
1 E E E
AAA, avidin-HRP (21
wash (4)
biotinylated well (3)
v
r-i
enzyme activity determination
r
(5)
Figure 1. A schematic diagram of the microtiter plate binding assay principle: (1) receptor a-sites bind with labeled ligand aBgt-biotin; (2) added avidin-HRP titratesthe remainingfree am-biotin; (3) biotinylated well extractsthe excess added avidin-HRP; (4) assay solution is washed off; (5) substrate is added to bound enzyme.
was added (finalconcentration 0.2 nM) and incubated for 1more hour. In the second set of samples for determination of the nonspecific binding, 50 pL of receptor was first allowed to bind with 50 p L of carbamylcholine (final concentration 5 X lW3 M) for 30 min and then incubated with 50 pL of aBgt-biotin for 2 h. Avidin-HRP was finally added into the sample followed by another hour of incubation. Because avidin-HRP binding to biotin-BSA on the solid support is much slower than that to aBgt-biotin in the solution,1°these two interactions are roughly noncompetitive. Displacement experiments were conducted in the same way except that the nAChR was first incubated with 50 pL of the analyte in place of carbamylcholine, and the incubation time was extended by 30 min to 1 h. Enzyme Activity Determination. After sucking all of the solution out of the wells, the ELISA plate was washed five times with PBS. The enzyme reaction was then started by adding 100 pL of TMB and 50 pL of H202 (0.01%) into the well. The substrate solution was incubated at room temperature with shaking. After 20 min, 50 pL of 0.2 N H2SO4 was added to stop the reaction, and the absorbance at 492 nm was measured with a microtiterplate reader? This detectionwavelengthwas chosen because it yielded the largest change in absorbance among the filter wavelengths available on the plate reader. Unless stated otherwise, all assay sampleswere run in triplicate, and the results were averaged to obtain the reported value. The enzyme activity varies somewhat from day to day even with the same amount of avidin-HRP bound onto the wall. Therefore, to standardize the data, results are expressed as % (percentage) enzyme activity. The value for 100% enzyme activity in each experiment was obtained by incubating 0.2 nM avidin-HRP PBS-BSA solution in the biotinylated microtiter plate under the same experimental conditions.
RESULTS AND DISCUSSION Biotinylation of ELISA Wells and Its Binding with Avidin-HRP. A standard protocol was employed to coat the plate without further optimization. Alarge excess of BSA (0.001%) was used to speed up the coating process. By controlling the incubation time, only a fixed amount of biotinBSA is immobilized on each well, which can be quantified (10) Takeuchi, T.; Rechnitz, G. A. Bioconj. Chem. 1990,1/4,227-230.
9020
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 14
12 10
8 6
1
0
2
I
4
-1 aBgt-blotln] ( n M
4
2
0’ 1 2 6 8 10 12
0.0
4
0
-’
avldln-HRP ( x i 0
0
mol)
Figure 2. Blndlng of avMin-HRP to the ELISA plate. The MTP was coated wlth 0.001 % biotin-BSA by incubating overnlght, blocked with 1% BSA for 45 mln at room temperature, and Incubated wlth avMlnHRP for 1 h at 4 O C wlth shaking. 0.6
I
1
0.4 0.3 -40
-
0.1
0.0
A
0
I
I
I
I
I
1
2
3
4
5
I
I
I
I
1
4
6
8
10
12
ll[aBgt-biotin] ( n M - ’ ) Figure 4. Enzyme acthrity (%) vs [aBgt-biotin] curve for the determination of the amount of “bound”and “free”labeled Ilgands. Small (inset): obtained by fixing [avidin-HRP] at 0.2 nM while varylng
the amount of aBgt-biotin present In the solution. Large: reciprocal plot, relative acthrlty = enzyme actlvlty/maxlmum enzyme actlvlty (obtained from the samples when only 0.2 nM avidin-HRP was introduced) = (% enzyme acthrlty)/100. (0)Assay I, y = 0.944 0 . 7 8 4 ~(X) Assay 11, y = 0.947 0.243~.
+
0.5
0.2
I
2
20
a2
d
10
6
incubation time @) Figure 8. The effect of incubation time on uptake of avidin-HRP onto the blotinylated plate at 4 O C . [avMin-HRP] = 0.2 nM (2 X lo-” mol).
Percent (%) enzyme actlvlty = enzyme acthrlty/maximum enzyme acthrky (obtained from a 5-h Incubation).
by avidin-HRP binding. Excess BSA is then removed by aspirating the coating solution. Protein concentrations higher than 0.001% should be avoided so that excessive leaching does not occur.11 The biotin-binding activity of avidin-HRP and the doseresponse curve for inhibition of its binding to biotin-BSA by free biotin have been previously reported.10 The binding behavior of avidin-HRP with biotinylated wells was studied by incubating various amountsof avidin-HRP in a microtiter plate. As shown in Figure 2, the binding exhibits a saturable behavior for avidin-HRP above 6 X 10-14mol. The absorbance was limited to about 0.6 because of the amount of biotinBSA immobilized and the incubation time with substrate solution. In the range from 0 to 2 X 10-14mol, the quantity of avidin-biotin bound onto the solid support is proportional to the moles of avidin-HRP present in the solution. Therefore, 2 X lO-l4 mol, which is 0.2 nM when converted to concentration, was chosen for later studies as it afforded the greatest sensitivity. The incubation time dependence of avidin-HRP binding to the solid support is demonstrated in Figure 3. The amount of binding reached a maximum only after 5 h; however, at 1 h approximately 80% of maximum was achieved. The relatively long incubation time is required for the binding of avidin to biotinylated wells because of the heterogeneous nature of the interaction. Determination of the Bound and Free Labeled Ligands. In order to obtain the amount of “free”and “bound” (11) Campbell, A. M. Laboratory Techniques in Biochemistry and Molecular Biology-Monoclonal A n t i b o d y Technology; Elsevier Biomedical: Amsterdam, 1983; Volume 13.
+
labeled ligands, a percent enzyme activity vs [aBgt-biotin] curve was generated by fixing the avidin-HRP concentration at 0.2 nM while varying the amount of aBgt-biotin in the solution (Figure 4, inset). Thus as long as one knows the percent enzyme activity, the correspondingfree aBgt-biotin concentration in the assay sample can be determined from this curve. The results from the first set of experiments (see the ExperimentalSection)provide the “free”signals and thoee from the second set provide the “total”signals. The difference between the “free” and “total” signals is the “bound” signal. For the purpose of accurately calculating the aBgt-biotin concentration from a given response, the raw responses from the inset of Figure 4were linearized by means of two reciprocal plots (Figure 4, large). These plots have a form similar to those of Lineweaver-Burk.12 The difference between the two curves in Figure 4 is an interesting phenomena. The possible source of this difference is mainly due to the change in the experimental design when going from assay I to assay 11. In the first assay, avidin-HRP and aBgt-biotin binding reaches an equilibrium before an aliquot of the solution is added to the microtiter plate and incubated for 1 h. However, in the second case both the avidin-HRP/aBgt-biotin and avidin-HRP/immobilized biotin binding occurs simultaneously in the microtiter plate, with a total incubation time of just 1h. Thus the binding between avidin-HRP and aBgt-biotin may not reach a true equilibrium due to the presence of immobilized biotin on the microtiter plate, and consequentlythere will be more avidinHRP bound to the walls. As a result, the amount of avidinHRP bound to the microtiter plate is higher in assay I1 than in assay I. However, this difference does not in any way appear to compromise the results of either assay. Solid-Phase Competitive Binding Assay I (Assay I). Effectof Carbamylcholine Concentration. From previously published work? carbamylcholine was shown to be useful as a specific displacer to displace specific binding of the labeled ligand so that the labeled ligand could only bind nonspecifically with the receptor. Thus the carbamylcholine concentration must be high enough to displace all the specificbinding but not so high as to displace nonspecifically bound aBgt(12)Guilbault, G. G. Analytical UsesofImmobilized Enzymes,Marcel Dekker, Inc.: New York, 1984; Chapter 1. (13)Blanchard, S.G.; Quast, U.; Reed, K.; Lee, T.; Schimerlik, M. I.; Vandlen, R.; Claudio, T.; Strader, C. D.; Moore, H. H.; Rafbry, M. A. Biochemistry 1979, 18, 1875-83. (14)Taylor, P.;Brown, R. D.; Johnson, D. A. Cum. Top. Membr. Trawp. 1983, 18, 407-43.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
3021
Table I. Effect of Carbamylcholine Concentration on the Enzyme Activity % enzvme activitv ~~
[carbamylcholine] $4)
assay I
assay I1
0 0.0005
100
100
0.006
98
48
0.05
41
79
Table 11. Interaction between nAChR and Avidin-HRP % enzyme activity lnAChRl (nM) wlcentrifwation w/o centrifugation 0.2 0.4 0.8
93
90
101 100
87
98
biotin; this concentration range was demonstrated to be 0.1 M. In this system, we found that when the to 1 X carbamylcholine concentration was above 5 X M, the enzyme activity decreased significantly (Table I). The experiment was conducted by incubating avidin-HRP (0.8 nM) with carbamylcholine for 2.5 h in teet tubes and then transferring it into the microtiter plate for another hour. Therefore, a carbamylcholine concentration of 5 X 10” M was chosen in assay I for displacement of uBgt-biotin from the specific binding sites. The possible reasons responsible for the observed decrease in enzyme activity are that carbamylcholine inhibits HRP activity,hinders avididbiotin binding, or displacesthe biotinBSA coated on the solid support. The fvst possibility was investigated by incubating avidin-HRP with and without carbamylcholine in cuvettes for 2 h and then determining the absorbance through a spectrophotometer. The results indicate that carbamylcholine does not affect HRP activity. As a matter of fact, the observed phenomena is mainly due to the displacement of biotin-BSA from the walls of the microtiter plate, which is proved in later studies. Consideration of Possible Nonspecific Interactions. Nonspecificinteraction of both the nAChR and avidin-HRP with the biotinylated plates proved to be negligible with an incubation time of 4 h. The avidin-HRP activity, however, could be affected by the presence of nAChR preparation, which was studied by two parallel experiments. In the fist set of experiments the supernatant of the nAChR preparation was centrifuged (27000g, 10 min) and the supernatant was used, while in the second case the crude nAChR preparation was employed directly without centrifugation. These two samples were incubated with avidin-HRP in test tubes for 1h and then in a microtiter plate for another hour. In Table 11,the enzyme activity shows decreasing trends in both cases, which is due to the nonspecificinteraction between the nAChR preparation and avidin-HRP so that less free avidin-HRP is available to bind with the plate. The result suggests that avidin-HRP nonspecifically interacta not onlywith the nAChR membrane, but also with the biological matrix present in the supernatant after centrifugation. However, the magnitude of this interaction is not significant even for the concentration of 0.8 nM used in all other studies if no centrifugation is employed. On the other hand, the enzyme activity difference between the samples with and without centrifugation is significant. The lower enzyme activity bound to the microtiter plate when the receptor preparation is centrifugedis likely due to adsorption of avidin-HRP to the walls of the test tube, which was used to incubate the avidin-HRP with the supernatant, before pipetting an aliquot into the microtiter plate. Avidin-HRP adsorption does not occur in the test tube if the receptor
40
’
0
I
I
20
40
I 60
I
80
I
I
I
I
100 120 140 160
incubatlon time (mln) F l g w 1. Optlmlzetkn of incubatlon time of aWn-HRP/free aBgtbktin bkrdkrg after the receptor ha8 been equilibrated wtth carbam ylcholine~and uBgt-blotin. [nAChR] 0.8 nM, [carbamylchollne] = 5 X 109 M, [aBgt-blotln] = 0.5 nM. (X) nAChR aBgt-bktin; (0) nAChR carbamyichoiine aBgt-blotln. Selected incubatkn time 60 min.
+
+
+
preparation is not centrifuged, owing to the presence of a high concentrationof proteins and surfactants in the receptor matrix. Optimization of the Incubation Times. Figure 5 shows the effect of incubation time on the avidin-HRP/aBgt-biotin binding after the receptor has been equilibrated with either uBgt-biotin or both carbamylcholine and uBgt-biotin. The percent enzyme activity difference obtained from two sets of experiments has almost reached a maximum after 60 min; this was fued as the incubation time for later experiments. The question of whether a significant amount of binding occurs between avidin-HRP and nAChR/uBgt-biotin complex was addressed by a kinetic study. An excess amount of nAChR (0.8 nM) was mixed with uBgt-biotin (0.1 nM), and then avidin-HRP was added to the mixture and incubated for different periods of time. The result shows that at up to 2-h incubation, enzyme activity is only decreased about 5% ; thus, the rate of formation for the nAChRIuBgt-biotin/ avidin-HRP complex is negligible. This is desirable because it demonstrates that avidin-HRP effectivelyonly binds with free aBgt-biotin in the aqueous phase and the biotin-BSA on the solid support. Therefore a step to separate free from bound uBgt-biotin is not needed to perform this w a y . Once the assay sample has been pipetted into a biotinylated well, Figure 3 illustrates that the maximum binding between the avidin-HRP and the solid support occurs after 5 h. However, because of the fact that the longer the incubation time, the more dissociation of the aBgt-biotin/ avidin-HRP complex and association of avidin-HRP with nAChR/uBgt-biotin, a 1-h incubation time was chosen for this step. Saturation Curve and Scatchard Plot. Figure 6 depicta typical curves obtained from assay I when varying the concentration of aBgt-biotin. The enzymeactivitydifference between sets 1 and 2 is due to the specific binding of the nAChR and uBgt-biotin, while that between sets 2 and 3 is due to the nonspecific binding. Using the relationship shown in Figure 4,all the percent enzyme activitywas converted into the correspondingamount of “free” and ‘bound” labeled ligand. Thus the saturation curve and Scatchard plot could be generated (Figure 7). From the Scatchard plot, the apparent dissociation constant WBB determined to be 9.4 X 10-11 M, and the maximum uBgtbiotin binding sites was 19nmol/g protein or 0.77 nM of aBgtbiotin binding sites. Comparing these values to those from a previous study? the & is around 1 order of magnitude lower, which may partly arise from the binding affiiity difference between uBgt-biotin, used in this work, and uBgt-
3022
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23,DECEMBER 1, 1992 100 80
U
a
m a9
60
40 20
0
1
2
3
4
5
6
-11.10-9 - 8 - 7
[~Bgt-biotinl[nMl Figure 6. compewhrebinding assay I cvv88obtahedby fixing [nAchR] at 0.8 nM, [carbamylcholine] at 5 X lo3 M, [avidin-HRP] at 0.2 nM, and varying the concentratbn of aBgt-biotin: (A)set 1, nAChR aBgt-biotin; (0) set 2,nAChR + cerbamylchollne aBgt-blotln; (X) set 3, aBgt-biotin only.
+
+
250 200 150 100
50
0
I
I
I
5
10
15
20
Bound (nmoilg protein)
Flguro7. Scatchard analysis for assay I. Same conditions as Flgure 6. Small (Inset): Saturation curve. Large: Scatchard plot. From the plot, & = 9.4 X lo-“ M, and S,= 19 nmol/g of protein = 0.77 nM of aBgt-biotin sites.
FITC, used in the previous work, with the receptor. The apparent dissociation constant of aBgt-biotin compares very favorably with the range of values reported in the literature (10”J-10-12)for aBgt itself, which indicates that the activity of aBgt-biotin is very similar to that of aBgt. Dose-Response Curves. Doseresponse curves for aBgt and cholinergic ligands were obtained (Figure 8) by using a labeled ligand concentration of 0.5 nM which results in the maximum percent enzyme activity difference between experimental sets 1and 2 (see Figure 6). Apparent dissociation constants can be subsequentlycalculated using the ICs0 values obtained from the displacement curves. These data are consistent with literature values using radiolabeled receptorbased assays and show lower detection limit than the fluorophore-labeled nAChR assay (Table 111). The reproducibility of the dose-response curves proved to be satisfactory, with no more than 20% error at the 90% confidence interval (N= 6 ) . The main sources of error are likely to be from the irreproducibility of the biotin-BSA coating from well to well and the accumulation of deviation caused by the multiple incubation steps involved. Solid-Phase Competitive Binding Assay (Assay 11). Effect of Carbamylcholine Concentration. The most difficult problem encountered in the development of the assay I1 method was the significant reduction of the enzyme activity bound to the plate even if a carbamylcholineconcentration as low as 5 X lo4 M was used (Table I). This is well below the concentrationof carbamylcholine needed to be an effective specific displacer. From the discussions above, we were
-6 - 5 - 4 -3 - 2 --1
0
1
2
log[anaiyte] (M) Figure 8. Dose-responsecurves of agonists and antagoniets to the a-subunttsof the nAChR (assay I). [nAChR] = 0.8 nM, (aBgt-bktln] = 0.5 nM, and [avidin-HRP] = 0.2 nM. Percent (%) bound (specmC binding) = bound/mdmum bound (obtained form the assay eempks when no dieplacers were Introduced): (A)aBgt;(0)pancuronlum; (+) ~~(~)Slalamine;(X)cerbemyldrdlne;(O)decemahonknn; ( 0 )bxamethonium. Small (inset): reproducibility of cerbamylcholhre curve. Error bare (90% confidence interval)were obtained for 6 Mais.
certain that carbamylcholine did not obstruct the activity of HRP. It was suspected that this effect was most probably caused by the displacementof the biotin-BSA adsorbed onto the solid support, and the magnitude of this displacement would increase with the incubation time. In w a y 11, carbamylcholine was allowed to stay in contact with the microtiter plate for 3.5 h instead of only 1h as was the case in assay I, which explains why the displacement was greatly increased in assay I1 even though the carbamylcholine concentration was 5 X 10-3 M in both cases. In order to solve this problem, the StabilCoat immunoassay stabilizer was utilized as asubatitute for the 1% BSA solution in the blocking step of the biotinylation procedure.16 The use of StabilCoat returned the amount avidin-HRP activity to 98% for the 3.5-h incubation; this is similar to the values obtained with the 1h incubation time used in assay I. This experiment also eliminated the possibility that carbamylcholiie hinders avidin-biotin binding. The StabilCoat ingredient interads with active biomolecules immobilized or adsorbed onto a surface and maintains their biological activity under extreme conditions such as high surfactant levels and high temperatures. It can be used to replace the blocking step in immunoassays, and coated ELISA plates retain nearly 100% activity for long as 5 months at 40 “C. Scatchord Anulysis. Under assay I1 experimental conditions, Figure 9 depicts the typical curves obtained when a f i e d amount of receptor is incubated with various concentrations of aBgt-biotin. The amount of ‘bound” and “free* were determinedfrom Figure 4 according to the correaponding percent enzyme activity, and from these data both the saturation curve and the Scatchard analysis were constructed (Figure 10). The & value obtained from the Scatchard plot is 9.6 X l@ll M which is almost the same as that from assay I. B , is 23 nmoVg of protein, Le., 0.91 nM of arBgt-biotin binding sites, which is also close to the result from assay I and well within the range of experimental deviation (the average concentration from assays I and I1 is 0.84 nM aBgt-biotin sites). However, because the assay was conducted directly in amicrotiter plate, the procedureis easier and more convenient for the purpose of automation; as an added bonus the time needed to perform the assay is shortened by 1h. Dose-Response Curves. Figure 11gives the dose-response curves of aBgt and various cholinergic ligands obtained using (15)StabilCoat. Immunoassay Stabilizer Product Information; BioMetric Systems, Inc.: Eden Prairie, MN.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
8028
Table 111. Apparent Dissociation Constants of Agonists and Antagonists Obtained from Assays I and I1 RRA (lit.) assay I
anal* ffBgt
7.4 x 6.6 X 5.3 x 3.3 x 2.6 X 8.4 X 2.1 x
carbamylcholine d-tubocurarine pancuronium gallamine decamethonium hexamethonium
assay I1 9.4 x 6.7 X 4.7 x 3.3 x 3.0 X 8.4 X 1.9 x
10-10
lW7 10-7
10-8 10-8 10-1 10-4
ref 13
ref 14
10-10
lo1 lo-'
1.2 x 10-7 2.0 x 10-7
10-8
10-6 10-1 lo-'
1.8 X 10-1 1.2 x 10-4
I
0
1
2
4
3
5
6
I W t - b i o W (nM) Flgure 8. Competittve binding assay I1 curves, obtained under the same conditions as Figure 6; (A)set 1, nAChR aBgt-biotin; (e)set 2. nAChR carbamyichoiine aBgt-biotin; (X) set 3, aBgt-biotin
only.
+
+
+
300
250 200 150 100
50 0
0
5
10
15
20
25
B o u n d (nmoi/g protein) Flgure 10. Scatchard analysis for assay 11. Small (inset): saturation curve. Large: Scatchard plot. From the plot, & = 9.6 X lo-" M, and S,= 24 nmol/g of protein = 0.91 nM of a-subunit.
assay 11. The apparent dissociation constants are calculated from the IC@ obtained from these curves (Table 111) and are almost identical with those calculated using the data from assay I. The deviation associated with the method is relatively large compared with that from assay I, showing a maximum error of 30% at 90% confidence interval (N= 6). The procedures of the two assay methods discussed in this paper are less complicated in both procedure and instrumentation compared to the previously reported FRA.g With
4.8 x 3.8 X 2.3 X 1.1x 8.3 X
10-7
10-1 10-8 10-6 10-1
FRA (ref 9) 9XlO-B 3Xlod 8XlO-B 3 X 1W7 4XlO-B BXlod 2x104
100 7
- 1 1 1 0 - 9 - 8 - 7- 6 - 5 - 4 - 3 - 2 - 1 0 1
2
log[analyte] (M) Flgurr 11. Dose-response curves of agonists and antagoniststo the a-subunits of the nAChR (assay 11).same conditions given for Flgwe 8: (A)aBgt;(0)pancwonium; (+) &tubocurarine; (e) gallamine; (X) carbamylcholine; (e)decamethonium; (0)hexamethonium. Small (inset): reproducibility of carbamylcholine curve. Error bars (90% confidence interval) were obtained for slx trlals.
the relatively short incubation time and the semiautomated capabilities of microtiter plate technology, screening of large numbers of cholinergic compounds can be easily conducted. The utilization of a solid phase for the complete separation of labeled ligand from the biological matrix simplifies the assay protocol and reduces the background signal, thereby enhancingthe sensitivity. The methodology is new and quite practical for the determination of agonists and antagonista of the nicotinic acetylcholine receptor. Moreover, use of avididbiotin labeling system should be easily adaptable to other receptor assays. To further simplify the assay protocol, solubilized acetylcholine receptors are expected to be directly immobilized onto the microtiter plate either by hydrophobic interactions or by covalent bonding. Alternatively, if a fluorescence microtiter plate reader, instead of a W/vis plate reader, is employed, fluorescence labels can replace the avidinbiotin system so that the assay format will become even less complicated.
ACKNOWLEDGMENT We gratefully acknowledge the support of NIH Grant GM25308. RECEIVED for review May 19, 1992. Accepted September 21, 1992.
