Anal. Chem. 1984, 56,801-806
peak current obtained from the old cells is attributed to the decrease of CoA content of the cell wall. As shown in Figure 5, the peak potential of the eluent, which is similar to that of CoA, was more positive than that of the whole cells. The transport characteristic of CoA to the electrode surface is estimated from Figure 4 for the two cases of free and cellbound CoA. The increasing slope of current obtained from the free CoA with time is about 5 times the decreasing slope of the current from the bound CoA. This result shows the diffusion of CoA being present in the cell wall is lower than that of CoA in solution. Therefore, the peak potential of whole cells is higher than that of CoA solution and the eluent from sonicated cells, and the peak current obtained from whole cells is also lower than that of CoA solution and the eluent. The microbial detection method described here differs in principle from the previous electrode system (5-7). Because the platinum anode of the previous electrode systems was controlled a t 0.20-0.35 V vs. SSCE and the peak current of this system was obtained a t 0.74 V vs. SSCE. Oxidation of CoA does not occur a t 0.20-0.35 V vs. SSCE. Moreover, the current from the previous system was affected by the concentration of dissolved oxygen, whereas the oxygen did not affect the peak current and potential in this system. The novel concept described here for determining cell number of S. cereuisiae lays the ground work for the devel-
80 I
opment of methods detecting microbial strains. Further developmental studies in our laboratory are now directed toward determining and recognizing various species of microorganisms. Registry No. CoA, 85-61-0.
LITERATURE CITED Postage, J. R. "Methods in Microbiology"; Norris, J. R., Ribbons, D. W., Eds.; Academic Press: New York, 1969; Vol. 1, pp 611-621. Hadiey, W. K.; Senyk, G. "Microbiology-1975"; Schlesslnger, D.,Ed., American Society for Microblology: Washington, DC, 1975; pp 12-21, Zafari, Y.; Martin, W. J. J. Clln. Microbiol. 1977, 5 , 545-547. Wilkins, J. R.; Young, R. N.; Boykin, E. H. Appl. Environ. Microbiol. 1977, 35, 214-215. Matsunaga, T.; Karube, I.; Suzuki, S. Anal. Chim. Acta 1978, 98, 25-30. Matsunaga, T.; Karube, I.; Suzuki, S.Appl. Mviron. Microbiol. 1970, 37, 117-121, Matsunaga, T.; Karube, I.; Suzuki, S. Eur. J . Appl. Microblol. Biotechno/. 1980, 70, 125-132. Stadtman, E. R.; Novelll, G. D.; Lipman, F. J. Biol. Chem. 1951, 797, 365-376. Leduc, P.; Thevenot, D. H. Bioelectrochem. Bioenerg. 1974, 7 , 96-107. Blaedei, W. J.; Henklns, R. A. Anal. Chem. 1974, 46, 1952-1955. Blaedel, W. J.; Jenkins, R. A. Anal. Chern. 1975, 47, 1337-1343. Dryhurst, G., Ed. "Electrochemistry of Biological Molecules"; Academic Press: New York, 1977; pp 365-389.
RECEIVED for review September 9,1983. Accepted January 11, 1984.
Potentiometric Digoxin Antibody Measurements with Antigen-Ionophore Based Membrane Electrodes M. Y. Keating and G . A. Rechnitz*
Department of Chemistry, University of Delaware, Newark, Delaware 19716
We describe and Illustrate a technlque, potentlometrlc ionophore-modulation Immunoassay (PIMIA), for the measurement of antibodies with conlugate based membrane eiectrodes. Fundamental operatlng varlabies for the technique are examined and demonstrated for the case of antibodies to the cardlac drug dlgoxln. Detection ilmits In the pg/mL range, with high seiectivlty over other antibodies and proteins, are readlly attalned. Through a competttlve blnding approach, the selective measurement of digoxln itself is also shown to be possible with this technique.
Antigen and antibody measurements are of importance in clinical chemistry, physiology, and modern biotechnology. Considerable effort has been made to develop potentiometric membrane electrode sensors for this purpose, but contrary to the more straightforward enzyme-immunoassay techniques where a potentiometrically measurable product is liberated (1-4),it is much more difficult to couple antibody-antigen reactions to membrane electrodes. In this paper we attempt to lay the foundation for a class of potentiometric membrane electrodes which respond to specific antibodies through modulation of a background potential fixed by a marker ion and to illustrate this concept with the development of a selective electrode for antibodies to digoxin, a steroidal cardiac drug. For lack of an established descriptive term, we call this technique potentiometric ion0003-2700/84/0356-0801$01,50/0
ophore-modulation immunoassay (PIMIA). The principle of the method is simple. An antigen or hapten corresponding to the antibody to be measured is chemically coupled to an ionophore to form an antigen-carrier conjugate. The conjugate is incorporated into a plastic support membrane and that membrane is mounted in the sensing tip of a conventional potentiometric membrane electrode (Figure 1). The resulting electrode is exposed to a constant activity of a marker ion chosen for its compatibility with the ionophore portion of the conjugate, under conditions which produce a stable and reproducible background potential. When an antibody capable of binding the antigen portion of the conjugate is added to the background electrolyte, a potential change (hE) proportional to the antibody concentration is produced. It will be shown below that other antibodies or nonspecific proteins cause negligible interference with the determination of the primary antibody. In the present study, the selectivity and high affinity of digoxin antibodies raised in rabbits for the drug are especially favorable since the intrinsic affinity constant has been reported to be 1.7 X 1O1OpUl-l(5). Moreover, the ionophores benzo-15-crown-5and cis-dibenzo-18-crown-6 have excellent solubility in the poly(viny1 chloride) support membranes employed. Although we have previously reported on some of the construction details (6) and preliminary analytical limits (7) of earlier electrodes, the present paper, detailing a full investigation of the newly studied digoxin antibody electrode system, represents the first comprehensive effort to interpret 0 1984 American Chemical Society
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e( ” o
Figure 1. Schematic and pictorial diagram of digoxin antibody sensing electrode: (a) PVC membrane containing digoxin-carrier conjugate; (b) inner filling solution, 0.01 M KCI; (c) plasticizer, dibutyl sebacate; (d) digoxin antibodies.
potentiometric responses in terms of membrane and solution variables.
EXPERIMENTAL SECTION Apparatus. Potentiometric measurements were made with a Corning Model 12 pH/mV meter and recorded on a HeathSchlumberger SR 204 strip chart recorder. All measurements were made in twin cells thermostated at 25 “C with a Haake Model FS temperature controller. Membranes were assembled in Orion Research 92 series electrode bodies. Orion 90-01 single junction reference electrodes with potassium chloride-agar salt bridges were employed and potential readings were taken with the antibody electrode inside a Faraday cage for optimum potentiometric stability. Reagents and Materials. All chemicals used were of analytical reagent grade. Deionized water was used throughout. Digoxin antibodies were obtained from Miles Laboratories (Elkhart,IN) as rabbit anti-digoxin-bovineserum albumin antisera (65-855; lot 5552, titer 1:25000,K, = 2.0 X 10’O M-l; lot 5553, titer 1:20000, K , = 2.7 X 1O1O M-’). With the information provided by the manufacturer, the respective digoxin antibodies concentrations were estimated by the use of Scatchard’s equation, r/c = nK, - rK,, at 50% binding of antibody sites ( n = 2, r = l), to be 0.22 mg/mL and 0.26 mg/mL. (The term r corresponds to the moles of hapten bound per mole of antibody at a free hapten concentration c, n equals moles of hapten bound at binding site saturation, and K , is the average intrinsic association condtant.) BSA antibodies were supplied (Miles) as rabbit anti-bovine serum albumin in lyophilized form (65-111, lot R725, titer 2.6 mg/mL). Normal pooled rabbit serum (64-291, lot 0015) and rabbit albumin (82-451, lot 42, fraction V, 99% pure) were also purchased from Miles. Rabbit y-globulins (G 0261, Cohn fraction 11, 99% pure) were obtained from Sigma (St. Louis, MO). Preparation of Digoxin-Benzo-15-crown-5 Conjugate. The ion carrier, benzo-15-crown-5, was modified by the introduction of an amino group at the 4‘ position via nitration (8) and catalytic hydrogenation (Figure 2). To the mixture of 2.5 g of benzo15-crown-5 (PCR Research Chemicals, Inc., Gainsville, FL) in 35 mL of chloroform and 30 mL of acetic acid, the nitrating solution 1.25 mL of concentrated HNO, (d = 1.42,70%)in 3.5 mL of acetic acid was added dropwise over 30 min. The reaction mixture was neutralized with aqueous K&03 and the chloroform layer sepa-
0)Q8YD
?
U
Flgure 2.
Scheme for preparation of digoxin-benzo-15-crown-5
conjugate.
rated. The aqueous layer was extracted with chloroform and the combined chloroform extracts were washed with water till neutral and dried over MgSO,. After stripping off the chloroform, the yellow solid was recrystallized in ethanol twice to yield pure 4’-nitrobenzo-15-crown-5,2.2 g, mp 95.5-96.6 “C. NMR (in CDC13) 6 3.6-4.3 (m, 16 H), 6.87 (d, 1 H), 7.74 (d, 1 H), 7.91 (dd, 1 H). The nitro crown ether was reduced by hydrogenation with Raney Ni as the catalyst (9). The mixture of 4’-nitrobenzo-15crown-5 (2.0 g) in 50 mL of DMF and 1teaspoon of Raney Ni/H,O slurry (Aldrich, Milwaukee, WI; pore 50 pm, surface area 80-100 m2/g, pH 10) was hydrogenated in a Parr Series hydrogenator 40 psi) for 1.5 h. After the DMF was distilled off under reduced pressure, the brown oil solidified upon chillingand sitting. The brown solid was dissolved in hot 2-propanol, and petroleum ether (8) was used to precipitate the dark oil. Upon chilling, a white solid on the beaker wall, weighing 1.0 g, was obtained. NMR (in CDC1,) 6 3.46 (broad, -NH2), 3.75-4.09 (m, 16 H, -CH,-), 6.20 (dd, 1 H) 6.27 (d, 1 H), 6.74 (d, 1 H). The attachment of digoxin to the ion carrier was accomplished by the periodate oxidation method of Erlanger and Beiser (10 ) and the modified procedure of Butler et al. (5,11,12). As shown in Figure 2, the digoxin was oxidized by sodium periodate at the terminal digitoxose to form the dialdehyde derivative. The dito aldehyde was allowed to react with 4’-aminobenzo-15-crown-5 form a seven-membered-ring Schiff base compound, which yields the digoxin-benzo-15-crown4 conjugate after reduction with sodium borohydride. Digoxin (Sigma, St. Louis, MO) (102 mg, 0.13 mmol) was suspended in 8 mL of 95% ethanol. Four milliliters of freshly prepared 0.1 M NaI04 was added and stirring continued for 2 h. The mixture turned clear during the course of the reaction and then turned to a suspension again, which stayed clear when 1mL of water was added. The cleared mixture was run through a 1 mL volume of prewashed Dowex 1x4-100 (Sigma, 1.4 mequiv/mL, C1- form) anion exchanger column to remove the excess periodate anion. The periodate was monitored before and after the ion exchange step using starch-iodide paper. 4’-Aminobenzo-15-crown-5(36.8 mg, 0.13 mmol) was added to the oxidized digoxin solution and stirred for 1 h. NaBH, (45 mg, 1.19 mmol) was then added to reduce the Schiff base. The reduction was continued for 24 h. The mixture was run through another 1mL volume of prewashed Dowex 1x4-100 anion exchanger column
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
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(M+K)+
48
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I /I 20
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0
108
150
200
250
300
350
450
400
,
100
150
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250
386
350
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Figure 3. Positive-ion fast-atom-bombardment mass spectrum of K+. digoxin-benzo-15-crown4
Flgure 4. Positive-ion fast-atom-bombardment mass spectrum of digoxin-dlbenzo-18-crown-6 4- K'.
to remove excess borohydride anion. The borohydride was monitored before and after the ion exchange step with iodinepotassium iodide solution. After the solvent was removed with a rotatory evaporator, chloroform (2 mL) and methanol (1mL) were added to the residue. The NaCl from the anion exchanger precipitated and was removed by filtration. The condensed SUpernatant yielded 140 mg of gold solid. The crude digoxinbenzo-15-crown-5conjugate was purified by gel filtration chromatography with a Sephadex LH 20 (bed dimensions 2.2 X 42 cm, 1mL/min methanol flow rate). A total of 160 1-mL fractions were collected and fractions were pooled according to a plot of UV absorbance, A193, against eluant volume, milliliters. Digoxin-benzo-15-crown-5 (mol wt 1030) eluted at eluant volume 88-103 mL as compared to the molecular marker, vitamin B12(mol wt 1350), at eluant volume 78-92 mL. Thin layer chromatography in silica gel showed that the purified digoxin-benzo-15-crown-5 was a single spot with CHC13-CH30H (1:l) as solvent system and I, vapor and/or 6 N H2S04 as detectors. UV absorption maxima in concentrated HzS04are 475 nm (molar absorptivity, e, SOOO), 386 nm (16000), 316 nm (11000), 224 nm (19000), 193 nm (47000). The molecular weight was confirmed on a Kratos MS-50 mass spectrometer using fast atom bombardment ionization. The digoxin-crown ether is a potassium ion scavenger. A peak corresponding to the (M K)+ ion was found in the spectrum (Figure 3) with the most abundant monoisotopic mass of 1070. Preparation of Digoxin-Dibenzo-18-crown-6 Conjugate. The carrier, cis-diaminobenzo-18-crown-6, was prepared from dibenzo-18-crown-6 (Aldrich, Milwaukee, WI) following Feigenbaum and Michel's procedure (9),but the purification was accomplished by recrystallization in hot absolute ethanol. The purified white solid has mp 177-179 OC; NMR (in CDClJ 6 3.99 (m, 8 H, -CH,-), 4.10 (m, 8 H, -CH,-), 6.21 (dd, 2 H, S = 7.7 Hz, J" -- 2.7 Hz), 6.28 (d, 2 H, J" = 2.7 Hz), 6.71 (d, 2 H, J' = 7.7 Hz). The attachment procedure of digoxin to cis-diaminobenzo18-crown-6was similar to that of digoxin-benzo-15-crown-5. The conjugate prepared was purified by gel filtration chromatography with Sephadex LH-20 (bed dimensions and methanol flow rate eluted at eluant unchanged). The digoxin-dibenzo-18-crown-6 volume 82-96 mL, which indicated the monosubstituted conjugate was the major product. TLC in silica gel showed only a single spot with CHC13-CH3OH (1:l)as solvent and I, vapor and/or 6 M H2SO4 as detectors. UV absorption maxima in concentrated H2S04are 276 nm (molar absorptivity, E, 24000), 387 nm (44000), 315 nm (34000), 226 nm (50000),193 nm (56000). The molecular
weight of the monosubstituted digoxin-dibenzo-18-crown-6was confirmed on a Kratos MS-50 mass spectrometer. A peak corresponding to (M K)+ was found in the spectrum (Figure 4) with the monoisotopic mass at 1177. Preparation of Digoxin-BSA Conjugate for Inhibition Study. The conjugate was prepared and characterized by closely following the procedure of Butler and Tse-Eng (11). The digoxin to BSA ratio was 20 to 1. Construction of the Membrane Electrode. The conjugates were immobilized in 0.2 mm thick poly(viny1chloride) (PVC) film with plasticizer (6). The membrane films were cast with PVC powder (0.25 g), tetrahydrofuran (THF) (6.0 mL), dibutyl sebacate (plasticizer, 0.25 mL), and various amounts of conjugates (0.3-3.0 mg) in 48-mm culture dishes. In the case of digoxin-dibenzo18-crown-6, poorly soluble in THF, 2 drops of methanol were needed to dissolve the conjugate initially. Further reduction of membrane conjugate concentration was accomplished by dissolving a portion of the stock membrane and recasting with additional PVC and plasticizer. Disks of 3 mm diameter were cut out for placement into the Orion 92 series electrode bodies (Figure 1) employed with 0.01 M KCl internal filling solution. Before use, electrodes were conditioned in 0.01 M KC1 overnight, since the crown moiety of the conjugate is a K+ ion carrier. Subsequently, a pH 7.4 0.1 M Tris-HC1 working buffer, containing 3 X M KCl, was employed. Antibody Measurements. In order to eliminate any potentiometric contribution from changes in pH or ion activities, all antibody measurements were carried out in 0.1 M Tris-HC1 buffer (pH 7.4) with fixed K+ ion concentration at millimolar levels. All the sera and protein samples (1-3 mL) were extensively dialyzed (Spectropor 2 dialysis tubing, molecular weight cutoff (MWCO), 12000-14 000) against 4 x 1liter of the background solution to remove any interfering ions. The calibration curves for antibodies, normal pooled serum samples, and pure protein samples were constructed by adding the appropriate amounts of stock solutions to 2 mL of the background buffer. The AE values plotted represent the difference between the base line potential and the steady-state potentials established in the test solution. For the digoxin antibody measurements, it was found necessary to place the electrode in 0.1 M glycine-HC1 (pH 2.8) buffer briefly (
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DIGOXIN ANTIBODIES CONCENTRATION
Figure 5. Effect of digoxin-benzo-15-crown4 concentration in PVC membrane on net potential responses to rabbit digoxin antibodies in Tris-HCI buffer (0.1 M), KCI 3 X M: 11.7 pgldisk (0);0.3 pgldisk (0); 0.1 pgldisk (A).
5
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0
with various amounts of digoxin antibodies for 30 min. The digoxin-dibenzo-18-crown-6/PVCmembrane electrodewas then tested for immunoresponse with antibodies whose binding sites were saturated with the digoxin-BSA antigen.
RESULTS AND DISCUSSION It might be helpful in connection with Figure 1to imagine a system in which the conjugate containing membrane is perfectly symmetrical, the internal and external reference electrode elements are exactly matched, and the K+ activities in the solutions contacting the inside and outside of the membrane are identical. Under such idealized conditions, the measured potential would be constant at zero. If the specific antibody is now added to the sample solution, we would expect to see a potential change, AE, if the electrode is indeed functioning as an antibody sensor and a calibration curve could be constructed by measuring AE as a function of antibody concentration. The selectivity of the sensor could be checked by similarly measuring the effect of possible interferences and other experimental variables could be controlled. The “real” system described in this paper is very similar except that the starting potential, before specific antibody is added, is constant at some nonzero value. Upon addition of specific antibody under otherwise controlled conditions, AE values are obtained which can be used to construct a calibration curve. It will be shown below that the magnitude of the AI3 values can be controlled, within limits, by adjustment of the membrane composition and that the sensor has excellent selectivity for the desired antibody over other antibodies or proteins in general. In order for the sensor to have practically useful dynamic properties, it is important that the concentration of the marker ion, e.g., K+, and its carrier in the membrane, e.g., the conjugate, be sufficient to maintain an adequate exchange current density. The key parameters in the overall system can be studied by separating the experimental variables. A series of electrodes were prepared for each of the conjugates, digoxin-benzo-15-crown-5and digoxin-dibenzo-18crown-6, in which the concentration of the conjugate in the sensing membrane varied over the range of 0.1 to 11.7 pg per membrane disk. From Figures 5 and 6, for the respective digoxin-carrier cases, it is clear that the potentiometric response to digoxin antibodies increases dramatically as the conjugate level in the sensing membrane is lowered. The trend is similar for both conjugates, but the best AE values are obtained for the digoxin-dibenzo-18-crown-6 case. This crown carrier is known to have a larger and more suitable cavity for complexing potassium ion. Attempts to lower the conjugate level below 0.1 pg per disk yielded even greater potential
10
15
20 w l m l
DIGOXIN ANTIBODIES CONCENTRATION
Figure 6. Effect of digoxin-dibenzo-18-crown-6 concentration in PVC membrane on net potential responses to rabbit digoxin antibodies in Tris-HCI buffer (0.1 M), KCI 3 X M: 11.7 pg/disk (0);1.1 pg/disk (*); 0.3 pgldisk (0); 0.1 pg/disk (A).
I
101
0 m
2 T
.4 D
.6 .8 ID 12 1.4 uo/Dcsc N OF OlGOXN-CARRER N W C MEMBRANE
Figure 7. Effect of membrane conjugate concentration on electrode M K+ response time to 15.8 pg/mL digoxin antibody (0)and 3 X (A). Times shown are steady-state responses for membrane containing digoxin-dibenzo-18-crown-6 conjugate.
changes but with very poor reproducibility and sluggish responses. The relationship between membrane conjugate level and response time is evident from Figure 7. We have noted that these response times become substantially shortened when electrodes have been repeatedly exposed to antibody and antigen containing solutions. These observations should be viewed in conjunction with Figures 8 and 9 where the response of the same series of electrodes to the marker ion, e.g., K+, is presented. All of the electrodes function as potassium ion sensors, but the response slope and linear range decline a t the lowest membrane conjugate levels exactly where the antibody response is greatest. Separate experiments carried out to evaluate the effect of buffer used to control solution pH and ionic strength showed that the Tris-HC1 buffer (0.1 M) a t pH 7.4 has a negligible effect on the observed potential. It is interesting to note that the crown compounds remain good K+ ion carriers even when conjugated to the large digoxin antigen. These observations suggest that the experimentally obtained antibody responses may arise from the reversible binding of the primary antibody to the antigen portion of the conjugate molecules available at the membrane/solution interface. Such an effect would be expected to be greatest when
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
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.
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Figure 8. Potentiometric response of digoxln-benzo-1 Bcrown-B/PVC membrane to K+ ion in Tris-HCI buffer (0.1 M, pH 7.4). Concentratlons 11.7 pg/disk of the digoxin-carrier are as foliows: 27.7 pg/disk (0); (A);0.3 pgldisk (W); 0.1 pg/disk (*).
5
0
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DIGOXIN ANTIBODIES CONCENTRATION
Figure 10. Electrode responses to digoxin antibodies alone (A)and to digoxin antibodies preincubated with 20 p g of digoxin-BSA (”).
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100 80
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I
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SAMPLE VOLUME ADDED -3
-2
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IW(K+)
Flgure 9. Potentiometric response of digoxin4ibenzo-18crown-6/PVC membrane to K+ ion In Tris-HCi buffer (0.1 M, pH 7.4). Concentrations 1.1 pg/disk of the digoxin-carrier are as follows: 11.7 pg/disk (0); (A);0.3 hg/disk (W); 0.1 pg/disk (*).
the concentration of conjugate molecules in the membrane phase is low, because a larger fraction of the conjugate sites can be bound by antibodies. Two critical experiments lend support to this hypothesis. Figure 10 shows a comparison of the electrode response for the 0.1 pg/disk case to digoxin antibodies alone and to antibodies which have been preincubated (summary of three trials) with the antigen, digoxin-BSA. In the latter case, no significant potential changes are observed over the entire range of antibody levels studied, as would be expected if the antibodies are no longer able to bind to the digoxin portion of the conjugate in the electrode membrane. This finding and the selectivity results detailed below (Figure 11) rule out the possibility of a mechanism involving simple protein adsorption. Since the antidigoxin antibodies were raised against digoxin-BSA immunogen, there is the possibility that anti-BSA
Figure 11. Comparison of responses of digoxin-dibenzo-18-crown-6 membrane (0.1 pgldisk) to digoxin antibodies (+) and to normal rabbit rabbit serum albumin stock solution (45 mg/mL) (A); rabbit serum (0); y-globulins stock solution (30 mg/mL) (0);BSA antibodies ( 0 ) .
antibodies may coexist with the primary antibodies. To ensure that such cross contamination is not contributing to the potentiometric response, separate “control” experiments (three trials) were carried out in which excess bovine serum albumin was added to the anti-digoxin antibody test solution. The AE values obtained in the presence of BSA were not different from those for anti-digoxin alone. It should also be noted (Figure 11)that anti-BSA antibodies produce no appreciable electrode response. The observed digoxin antibody responses are fully reversible and base line potentials are reestablished if the electrodes are returned to the background buffer after brief exposure to a pH 2.8 glycine-HC1 buffer. Lowering the pH evidently dissociates antibody-antigen binding and, in conjunction with electrode reconditioning in the original background buffer, serves to restore the membrane/solution interface. These observations also suggest that the very high molecular weight
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
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a
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-2
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FI ure 12. Potentiometric response to digoxin antibodies at varying K marker ion levels but constant membrane conjugate level (0.1 pg/disk): (0) and (A),digoxin-benzo-15-crown-5 with 19 pg/mL and 7.9 pglmL antibody, respectlvely; (0),dlgoxin-dibenzo-1&crown-6wlth
o+
B
7.9 pg/mL antibody.
antibody molecules do not enter the membrane phase. It will be noted that the addition of digoxin antibodies always produces a potential change in the direction of increasing apparent potassium ion activity. However, experiments carried out with conventional K+ selective membrane electrodes, e.g., Orion's 93-19 potassium ion electrode paired with 90-01 single junction reference electrode, showed no detectable changes in K+ activity when antibodies are added. Taken together, these findings indicate that we have neither a "titration" of K+ by the antibodies (which would result in potential changes in the direction of decreasing K+ activity) nor a release of K+ from the membrane. Finally, the electrode is shown (Figure 11) to have excellent selectivity for digoxin antibodies over other antibodies and proteins. An additional experiment (not shown) with anticortisol antibodies also gave negligible response. This is particularly significant since the digoxin antibodies used in this study were raised, according to the supplier, in rabbits immunized with digoxin-bovine serum albumin antigen. It is known (13-17) that human serum albumin binds to digoxin with an association constant of lo5 M-l and it has been shown that serum albumin from rabbit and other species bind to several cardiac glycosides (18). However, we found negligible interference of purified rabbit serum albumin on electrode response (Figure 11). The AE values obtained for the digoxin antibodies are routinely reproducible to k1.5 mV or better, although no special attempts were made to optimize the analytical precision for the purpose of this study. All of these experimental results are consistent with a model (Figure 1) in which the potential modulation results from selective and reversible binding of the digoxin antibodies to the digoxin portion of the conjugate molecules a t the membrane interface. We have considered and rejected a further hypothesis in which antibodies are competing with the K+ marker ions for the conjugate sites. If this hypothesis were correct, we would expect to see a continuing increase in hE values in response to antibody additions as the K activity is lowered. Figure 12 shows that this is not so and, instead, displays an ion activity profile with an optimal concentration of 3 x 10-3 M. The conjugate containing membranes retained their activity for a t least a month when stored a t room temperature prior to mounting. Fully assembled electrodes, stored in buffer between measurements, were routinely used for 1-2 weeks before membrane replacement. Since each membrane film casting is sufficient for the preparation of literally dozens of
1:o
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( ANTIPEN 1
Figure 13. Competitive binding assay for digoxin (0)using the digoxin-benzo-l5-crownd/PVC electrode (0.1 pgldisk) at constant (19.0
pg/mL) antibody concentratlon; cortisol (A).
electrodes, the cost of the membranes is a negligible factor. Although this paper has focused upon potentiometric antibody measurements, the PIMIA technique described has also been used in preliminary experiments for detection of the corresponding antigens in a manner comparable to that of conventional immunoassay. By use of the competitive binding approach, digoxin can be selectively measured with moderate sensitivity (Figure 13). The nonrelated steroid cortisol gives no reactivity even in large excess, while deslanoside (not plotted) showed the modest immunological cross reactivity expected for a closely related compound.
ACKNOWLEDGMENT We are indebted to Robert Cotter and David Heller of the Middle Atlantic Regional Mass Spectrometry Center at Johns Hopkins University for valuable assistance. LITERATURE CITED (1) Meyerhoff, M. E.; Rechnltz, G. A. Anal. Biochem. 1979, 95,483-493. (2) Gebauer, C. R.; Rechnitz, G. A. Anal. Biochem. 1982, 124, 338-348. (3) Alexander, P. W.; Maitra, C. Anal. Chem. 1982, 54,68-71. (4) Boltieux, J.-L.; Desmet, G.; Thomas, D. Clln. Chem . (Winston -Salem, N . C . ) 1979, 25, 318-321. (5) Smith, T. W.; Butler, V. P., Jr.; Haber, E. Biochemlstry 1970, 9 ,
331-337. (6) Rechnitz, G. A.; Solsky, R. L. U.S. Patent 4402819, 1983. (7) Keating, M. Y.; Rechnitz, G. A. Analyst(London) 1983, 108, 766-768. (8) Ungaro, R.; El Haj, E.; Smid, J. J. Am. Chem. SOC. 1976, 98, 5198-5202. (9) Feigenbaum, W. M.; Michel, R. H. J. Polym. Scl. 1971, 9 ,817-820. (IO) Erianger, E. F.; Beiser, S. M. Roc. Natl. Acad. Scl. U.S.A. 1964, 52,68-74. (11) Butler, V. P., Jr.; Tse-Eng, D. I n "Methods in Enzymology"; Langone, J. J., Van Vunakis, H., Eds.; Academic Press: New York, 1982: Vol. 84. (12) Butler, V. P., Jr.; Chen, J. P. Roc. Natl. Acad. Sc/. U.S.A. 1967, 57. 71-78.
0: S.; De Martlno, A. G. J. Clin. Invest. 1969, 4 8 , 1041-1053. Evered, D. C. Eur. J. Pharmacol. 1972, 18, 236-244. Voshall, D. L.: Hunter, L.: Gradv, H. J. Clin. Chem. (Winston-Salem, N . C . ) 1975, 21, 402-406. Shaw, W. Ciin. Chem. (Winston-Salem, N.C.) 1975, 21, 636. Holtzman, J. L.; Shafer, R . B. Clln. Chem. (Wlnston-Salem, N . C . ) 1975. 21. 636-637. Farah, A.'J. Pharrnacol. Exp. Ther. 1945, 83, 143-157.
(13) Lukas,
(14)
(151 . ,
(16)
(17) (18)
RECEIVED for review November 3, 1983. Accepted January 24,1984. This research was supported by NIH Grant GM25308. Portions of this work were presented a t the 10th Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies, Philadelphia, PA, September 27, 1983.
