1241
Anal. Chem. 1986, 58, 1241-1245
Both glass beads and silicon carbide boiling chips break up in the high-temperature fusion used in this procedure but the glass beads are free of metals that form insoluble fluotides. The silicon carbide boiling chips are not. Neodymium fluoride was preferred as a carrier over either cerium fluoride or lanthanum fluoride because neodymium is more soluble in the small pyrosulfate fusions used throughout this procedure. The lanthanum reagents seemed to have more radioactive contaminants in them than neodymium. However, if the neodymium or cerium reagents require purification, it can be done ( I ) . In the uranium, plutonium, and americium determinations, neodymium must be added to fluoride solutions to coprecipitate the nuclides. To do this, rapid mixing and slow precipitation are required. Precipitation before complete mixing must be avoided as much as possible. Neodymium precipitates much slower than either cerium or lanthanum. With only 50 bg of neodymium being added, the precipitation appears to be a homogeneous one. Precipitations of this kind can be more easily observed in a darkened room with a flashlight beam directed up through the bottom of the clear 50-mL roundbottomed polycarbonate centrifuge tube (Tyndall beam effect). The HT-100 filters used previously (6) are no longer available. However, the HT-200 filter, with twice the pore size, can be made to work as well if the substrate suspension is placed in a sonic bath for 15 min each day before use. This treatment probably breaks up the particles of neodymium fluoride and the finely divided precipitate plugs the pores in the HT-200 filter and helps it to have the apparent pore size of an HT-100 filter.
If a sonic bath is not available, HT-200 filters will work nicely if the vacuum is decreased to give a flow rate of 1drop/s through the filter. Nothing else needs to be changed. The recoveries ranged from 85% to 95%. The resolution for full width at half maximum (fwhm) was between 60 and 70 keV. Decontamination factors ranged between lo3 and lo4.
ACKNOWLEDGMENT The author wishes to thank K. W. Puphal for useful suggestions in the development of this procedure. The author also thanks R. L. Williams for the many a spectrometry measurements needed to complete this work. Registry No. DTPA, 67-43-6;neodymium fluoride, 13709-42-7; ceric iodate, 13813-99-5;thorium, 7440-29-1;uranium, 7440-61-1; plutonium, 7440-07-5; americium, 7440-35-9. LITERATURE CITED Bernabee, R. P.; Perclval, D. R.; Hlndman, F. D. Anal. Chem. 1980, 52, 2351-2353. Sill, C. W.; Puphal, K. W.; Hindman, F. D. Anal. Chem. 1974, 4 6 , 1725-1737. Filer, T. D. Anal. Chem. 1974, 4 6 , 608-610. Moore, F. L. Anal. Chem. 1983, 35, 715-719. Sill, C. W. Anal. Chem. 1974, 46, 1426-1431. Hindman, F, D. Anal. Chem. 1983, 66, 2460-2461. Puphal, K. W., Department of Energy, IDO-12096, 1982. Martin, D. B., private communication, Radiological and Environmental Sclences Laboratory, Department of Energy, 550 Second St., Idaho Falls, I D 83401, November 15, 1980.
RECEIVED for review August 12, 1985. Resubmitted October 8, 1985. Accepted December 24, 1985. Use of commercial product names is for accuracy in technical reporting and does not constitute endorsement of the product by the United States Government.
Drug Antibody Measurement by Homogeneous Enzyme Immunoassay with Amperometric Detection Celeste A. Broyles and Garry A. Rechnitz*
Department of Chemistry, University of Delaware, Newark, Delaware 19716
A homogeneous enzyme Immunoassay Is described for the determination of drug antlbodles. The method Is based upon inhlbitlon of the enzyme actlvity of an enzyme-antigen conjugate by the correspondlng antlbody that Is to be measured. The activity Is monitored by amperometrlc detection of fhe rate of NADH oxidation at a platinum electrode. The technique Is Illustrated by uslng the lldocalne-antl-lldocalne system and ylelds callbration curves at nanogram levels of antlbody with absolute sensltlvlty dependent upon the original amount of enzyme-antlgen conjugate. Interferences such as protein adsorption and antl-qulnidine cross-reactivity are shown to have mlnlmal effect on the assay, whlch has a precislon of 5%.
The determination of immunochemicals, both qualitative and quantitative, continues to engage researchers in diverse branches of chemistry and biology (1,2). Radioimmunoassay and advances in immunodiffusion techniques have increased the sensitivity of analytical methodology to detect antibodies 0003-2700/86/0358-1241$01.50/0
and antigens (3, 4). A more recent development, enzyme immunoassay (EIA) (5) has found wide application. Numerous variations of EIA, such as enzyme-linked immunosorbent assay (6)for determining both antibodies and antigens and enzyme multiplied immunoassay technique (EMIT) (7) for measuring antigens, have been described. Immunoassays are categorized as either heterogeneous, requiring a separation step during the analytical procedure, or the more desirable homogeneous assay, involving no separation step. While homogeneous EIA (HEIA) has been successfully employed for determining haptens (7), it has been less widely applied to antibody measurements, especially at the nanogram levels of interest. In this paper, we propose an electrochemical homogeneous enzyme immunoassay for drug antibodies using amperometric detection. Electrochemical homogeneous assays for antibodies have been reported using ion-selective electrodes with complement-induced lysis of liposomes (8)and sheep red blood cells (9) and using a Ag2S electrode (IO)to monitor denaturation of the protein. None of these assays was shown to be suitable at nanogram per milliliter concentrations, and all are rather 0 1986 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
complicated. Simpler methods have been reported (11,12) using chemically modified electrodes. These have met with limited analytical success and require further investigation. The procedure proposed here is designed to simplify current antibody determinations while achieving the required sensitivity. The reaction sequence is based upon antibody inhibition of the activity of an enzyme conjugated to an antigen bound by the antibody. This phenomenon is well-known and is employed in commercial kits for the determination of drugs (7) where the sample antigen and an enzyme-conjugated antigen compete for a limited amount of antibody. This principle has been adapted to various analytical systems and detection modes (13-15) including electrochemical detection of NADH at a carbon electrode (16,17). In our study, we have eliminated the competitive antigen aspect of the assay and applied the inhibition phenomenon to the determination of antibodies, demonstrated here with anti-lidocaine. The enzymatic system is that of glucose 6-phosphate dehydrogenase. The activity of the enzyme-antigen conjugate is monitored amperometrically. No separation step is necessary, and the technique is useful for antibody measurements at nanogram per milliliter concentrations with minimal interferences.
EXPERIMENTAL SECTION Reagents. Nicotinamide adenine dinucleotide (NAD, N7004, N0632), glucose 6-phosphate disodium salt (G6P, G7250), 6phosphogluconic acid (P7877),glucose-6-phosphatedehydrogenase (GGPD, L. mesenteroides G5760, G5885), and lidocaine (L-7757) were purchased from Sigma. NAD and G6P were reconstituted with water and the G6PD diluted in pH 8 glycine 0.1% bovine serum albumin (BSA, Sigma A4503). Lidocaine was dissolved in water made slightly acidic with HC1. The G6PD-lidocaine conjugate (G69D-L, which is not to be taken as a 1:l stoichiometric conjugate) was purchased from Syva Co. as part of the EMIT lidocaine assay kit (6M119, reagent B). It was reconstituted according to manufacturer specifications in distilled, deionized water. No information was available on the enzyme activity or antigen concentration of this conjugate. (Under assay conditions comparable to unconjugated enzyme, the G6PD-L was estimated to be 0.5 unit/mL.) Rabbit anti-lidocaine-BSA (lot RA3521F) and rabbit anti-quinidine-BSA (lot RA11505F) were products of Cambridge Medical Diagnostics, Inc. This company estimated the immunoglobulin to be 510% of the solution; therefore, a value of 0.1 mg/mL was used as a basis for subsequent calculations. Both antibodies were dialyzed against 0.1 M pH 7.4 Tris.HC1 and dilutions made with water. Tris buffer was made with Sigma Trizma Base (T-1503, tris(hydroxymethy1)aminomethane)and adjusted to pH 8 with HC1. Hydrogen peroxide, 30%, was purchased from Fisher and dilutions made with water. Distilled, deionized water was used throughout, and all reagents were stored at 4 OC when not in use. Instruments. Measurements of NADH and peroxide were made with a Yellow Springs, Inc., oxidase probe (Model 2510) and meter (Model 25). The probe consists of a platinum anode (1.5 mm diameter) surrounded by a silver cathode (7 mm diameter) with a polarizing voltage of 700 mV (nominal,nonadjustable). It is designed for the amperometric determination of H202. The probe was assembled according to manufacturer instructions. The tip is covered with a piece of collagen membrane (supplied by the manufacturer) secured around the tip with an O-ring, and the probe is then fitted into a plastic sleeve so as t o leave only the base of the tip in contact with solution. The collagen is recommended for protection of the electrodes and for defining the diffusion path to the tip. Reactions were carried out in a glass cell, thermostated at 30 "C with a Haake temperature bath. The amperometric response was followed on a Heath strip chart recorder and initial rates calculated manually from recorder tracings. Peroxide was measured by the change in current (nA) from base line to peak read from the meter as well as the chart paper. Peroxide Measurement. After the probe attained a steady base line in 3 mL of Tris buffer, 10 pL of 0.03% H202was injected into the cell. The peroxide readings were used as an independent measure of stability of the Pt electrode. If a decrease of signal
S + E-Ag
+ Ab
+ P
+
E-Ag + E-Ag:Ab
(11)
inactive Flgure 1. Equations for an enzyme immunoassay where the enzyme activity of an enzyme-antigen conjugate (E-Ag) is inhibited by antigen-antibody binding: (Ab) antibody, (S)substrates, (P) products; (I) blank, (11) Ab determination.
response was observed, it was interpreted as deterioration of the probe surface. A molecule larger than peroxide and less likely to cross a protein film could also have been used, but the peroxide test was sufficient for the intended purpose without the risk of further contaminating the probe. Anti-Lidocaine Calibration Curve. The probe was placed in the cell with a sufficient volume of Tris buffer, for a total of 2.1 mL after addition of all reagents, and allowed to reach base line. The appropriate reagents were then pipetted directly into the solution. The rate curves were followed for approximately 5 min and the initial rate calculated from the earliest linear portion after any lag phase. Two calibration curves were run covering two antibody concentration ranges. For the higher anti-lidocaine concentrations,an appropriate amount of the antibody was added to the buffer followed in quick succession by 60 pL of 0.035 M NAD, 40 pL of 0.15 M G6P, and finally 40 pL of G6PD-L to start the reaction. In the case of lower antibody levels, 50 pL of G6PD-L and various concentrations of anti-lidocainewere mixed together in a vial. The mixture was left at room temperature for 5 min; just prior to the end of this time, 70 pL of 0.035 M NAD and 50 pL of 0.15 M G6P were added to the Tris buffer (in the cell, equilibrated with the probe). The reaction was initiated by injection of the GGPD-L/anti-lidocaine mixture. Blanks contained no antibody. Interference Experiments. Investigation of several variables of the antibody assay followed the general procedure just outlined. Changes in the order of addition of reagents were sometimes necessitated by the particular aspect being studied. The concentration of the substrate, cofactor, antibody, and enzyme-antigen conjugate sometimesdiffered among the sets of experiments, but this was usually based on judicious use of reagents. Measurement of the KIMfor unconjugated G6PD (0.1 unit and 0.02 unit) was carried out in the presence of excess G6P (2.86 mM) and varying amounts of NAD (0.042-1.67 mM). The reciprocal of the initial rate was plotted vs. the reciprocal of the NAD concentration and linear regression of the straight line used to calculate the KM.
RESULTS AND DISCUSSION The basis of the immunoassay described here is shown in Figure 1. Increasing amounts of antibody bind more of the enzyme-antigen conjugate and will, to a certain degree, proportionately decrease the enzyme activity. The reaction used in this study is
+
+
-+ GGPD-Ag
G6P NAD" Ab 6-phosphoglucono-&lactone
G6PD-Ag:Ab (inactive)
NADH
+ H+ + Ab +
+ GGPD-Ag
(active) (1)
where Ab and Ag represent an antibody and its corresponding antigen, respectively. The G6PD activity is followed by the change in current produced by the oxidation of NADH. The electrochemistry of NADH and NADPH at electrode surfaces has been investigated (18, 19). Preliminary experiments showed that the oxidase probe gave a linear response to millimolar concentrations of NADPH. None of the reactants showed an electrochemical response at the Pt electrode, thereby ensuring a steady base line through to the point of reaction initiation and eliminating concern for interfering background rates. Likewise, the enzyme-antigen-antibody complex itself gave no signal. The lactone hydrolyzes to 6-phosphogluconate at pH 8. Samples of 6-phosphogluconic acid were not electroactive. Therefore, the oxidation of NADH
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
'1
I
a
l
b
provides a ready means of monitoring the GGPD activity in this reaction. The lidocaine-anti-lidocaine system was chosen to demonstrate this technique simply because of the availability of this pair of reagents, including an enzyme appropriate for the instrumentation of interest. The complexity of reaction 1 and possible factors influencing the inhibition process led to investigation of several experimental variables to develop a method with analytical utility. Procedural changes and potential interferences were studied and used to discern effects that might be reflected in the antibody assay. Following discussion of these studies, the calibration curve and precision data are presented. Reverse Addition of Antibody a n d Conjugate. The order of addition of reagents should affect neither the immunochemical reaction nor the inhibition if there are no unforseen factors contributing to these reactions. Normally, as stated in the experimental section, GGPD-L was added to the reaction mixture after anti-lidocaine was already in the reaction cell. Figure 2 illustrates the resulting rates when addition of these two reagents is reversed. The sequence was tested with addition of anti-lidocaine immediately after addition of GGPD-L (before any increase in base line could be measured) and with addition of anti-lidocaine after the enzymatic reaction was established for 1min. The curves clearly show that in either case, order of addition of these two reagents leads to similar inhibition of GGPD activity. Further, it is obvious that timing becomes a factor if the antibody is added after the enzyme reaction is in progress. There is only a short lag phase for this reaction; therefore, within 1rnin of adding the enzyme there is ample evidence of NADH oxidation. The difference in rate profiles in Figure 2 should be noted. When the enzyme reaction was allowed to proceed for 1min before antibody addition, curve d nearly overlaps curve a at the start, but after addition of antibody, the rate falls off. Within 5 minutes of antibody addition, the total change in nanoamperes for curve d is approximately equivalent to that of curves c and b where there was no delay before antibody addition. Incubation of Immunochemical Reagents. Antigenantibody reactions typically require incubation times of a half hour or more for full equilibration. Therefore, it seemed possible that increased inhibition would result if some such equilibration were used in conjunction with the procedure being developed. The GGPD-L was mixed with anti-lidocaine and allowed to react for 10 min before the enzymatic reaction was initiated. The conjugate and antibody were mixed in (a) a vial, at room temperature, separate from the cell and probe, and (b) in the cell with buffer, at 30 "C. In both cases the inhibition was significantly greater than that achieved without premixing (Figure 3). As will be shown, this increased inhibition of enzyme activity was used to advantage in constructing an anti-lidocaine calibration curve,
l
c
A
1 min
rnin
rnin
Flgure 2. Dependence of enzyme activity inhibition on order of reagent addition (0.048 kg/mL anti-lidocalne (Ab) and GBPD-L (E-Ag)): (a) no Ab, (b) E-Ag added immediately after Ab, (c) Ab added immediately after E-Ag, (d) Ab added 1'/, min after E-Ag (t). The t o for each reaction is marked below the curves (I).
,.
1243
Figure 3. Effects of incubation of 0.048 pg/mL anti-lidocaine (Ab) with G6PD-L (E-Ag) on enzyme activity inhibition: (a) no Ab, (b) no incubation of Ab and E-Ag before reaction initiation, 10-min incubation of Ab and E-Ag before reaction initiation (c) in the reaction cell and (d)
outside of the reaction cell. The t o for each reaction is marked below the curves (I). Table I. Effect of Albumin on Assay
rate of assay, nA/min
system
noBSAa
BSA
no anti-lidocaine 35 WLof anti-lidocaine 70 WLof anti-lidocaine
4.44
4.81 3.87 3.28
3.8 3.18
%RAD" 4
0.9 1.5
"BSA = bovine serum albumin; RAD = relative average deviation between pair of single measurements. BSA (0.4% final dilution) was added after NAD and G6P (0.83 mM and 2.8 mM final dilution, respectively) and before anti-lidocaine (0.005 mg/mL) and GGPD-L (30 pL); 8 min in Tris buffer wash between assays. Assay in Presence of Albumin. Antibody measurements are important on an industrial level to follow their progress during antibody production and in clinical laboratories to aid in diagnosis and treatment of disease. In the latter case, antibody may be measured in serum samples that have a high protein content. The assay being developed was, therefore, tested in the presence of bovine serum albumin (BSA) to approximate clinical conditions. The assay was performed, as usual, with and without addition of BSA. Results are given in Table I. While rates are slightly higher for all experiments that included BSA, all are within the precision of the technique. The large protein does not have a significant effect on the individual assays per se, but persistent exposure of high-protein samples to the probe requires precautionary measures. The proteins apparently adsorb to the collagen and can be removed by occasional 5-10-min wash in buffer. Results from three interference studies are summarized in Table I1 and discussed below. Values represent single measurements. Effect of Antibody on Unconjugated Enzyme. Large proteins, including the antibody itself, may exert nonspecific effects on the enzymatic reaction regardless of the immunochemical reaction. Unconjugated GGPD was incubated with anti-lidocaine to check for interferences such as steric hindrance by the antibody on the enzyme/substrate interactions. (Enzyme activities of approximately 0.06 unit and 0.01 unit, based on Sigma unit definition, were selected empirically to be similar to rates used in the various assays with GGPD-L.) With the higher activity of GGPD, the difference in rates (Table IIA) between the blank assay and that with anti-lidocaine is only 3.1% relative average deviation (RAD). No difference is evident between the blank and inhibition assays at the lower enzyme activity. Nonspecific effects of free lidocaine were similarly investigated and also showed no significant differences in rates at either level of GGPD activity. These results imply that the inhibition of the enzymatic rate
1244
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table 11. Effect of some Variables on Assay Results variable
assay rate, nA/min
A. Immunochemicals and Unconjugated G6PD 0.06 unit 0.01 unit
blank (no anti-L)" GGPD + 0.095 pg/mL anti-L premixed 5 min outside of cell before assay G6PD + 0.11 pg/mL lidocaine premixed 5 min outside of cell before assay
GGPD
GGPD
7.14
1.10
6.71
1.13
7.30
1.12
I
B. Anti-Quinidine Cross-Reactivity blank (no anti-L, GGPD-L) anti-quinidine (no anti-L) 0.095 fig/mL 0.95 fig/mL 0.048 pg/mL anti-L (no anti-quinidine) 0.048 pg/mL anti-L + 0.095 pg/mL anti-quinidine
2.54 0.2
2.69 2.42 1.77
1.80
C. Effect of Protein Adsorption
blank (no anti-L) 0.07 fig/&L anti-L with immediate addition of GGPD-L 10-min incubation of 0.07 pg/mL anti-L in cell before addition of GGPD-L 10-min incubation of GGPD-L in cell before addition of 0.07 pg/mL anti-L
1.54 1.18 1.04
1.06
anti-L = anti-lidocaine-BSA. plotted in Figures 2 and 3 is due to the immunochemical reaction and not a nonspecific side effect of the technique. Antibody Cross-Reactivity. Anti-quinidine was examined for cross-reactivity with lidocaine (Table IIB). (Quinidine, like lidocaine, is an antiarrhythmic drug.) The effect of anti-quinidine, at both 1pg/mL and 0.1 pg/mL, on the blank assay was minimal (2.4% RAD and 2.9% RAD, respectively). When anti-quinidine and anti-lidocaine were combined in the assay the resulting inhibition of the enzyme was essentially the same as that when only anti-lidocaine was used. Thus, anti-quinidine does not recognize lidocaine on the enzyme conjugate. In addition, the anti-quinidine, when viewed as an example of a large protein with the potential to interfere with this assay, appears not to have an adverse effect on either the enzymatic or immunochemical reaction. Protein Adsorption. A major concern while developing this assay was contamination of the Pt electrode due to protein adsorption. Therefore, the collagen-covered Pt was exposed to the immunochemical reagents to examine this problem. Anti-lidocaine was incubated in the cell with buffer and the probe for 10 min prior to initiation of the reaction, and, in another experiment, GGPD-L was similary incubated 10 min before the start of the reaction. Both experiments yielded lower rates than that of the usual anti-lidocaine assay having no preincubation of reagents (Table IIC). It is not clear what caused this behavior because HzOztests did not exhibit decreased current response as expected if protein adsorption or surface contamination of Pt had occurred. Perhaps, there was weak, but sufficient, adsorption on the collagen to restrict the movement of NADH. The 10-min incubation time used here is longer than the total assay time of previously described experiments in which little or none or the decrease in rate could be accounted for by other than antibody inhibition of the enzyme activity after binding to the antigen. Therefore, limiting the time the reagents are in contact with the probe diminishes adsorption problems. Anti-Lidocaine Calibration Curve. Having confirmed the feasibility of measuring the antibody via the inhibition assay, calibration curves for anti-lidocaine were established.
0.4
0.6
0.8
1 .o
pglmL anti-lidocaine Figure 4. Inhibition curves resulting from HEIA of anti-lidocaine with GGPD-L: (A) 60 pL of 0.035 M NAD, 40 pL of 0.15 M G6P, 40 pL Of G6PD-L, (B) 70 FL of 0.035 M NAD, 50 1L of 0.15 M G6P, 50 pL of G6PD-L with anti-lidocaine after 5-min preincubation.
Two experimental procedures (described previously) were followed for two different concentration ranges of antibody. The pair of complete inhibition curves is given in Figure 4. The more straightforward procedure (Figure 4A, single measurements) yielded a calibration curve with a linear range from 0.01 to 0.1 pg/mL. The slope was -16 with a coefficient of determination, r2, of 0.9823. Varying concentrations of antibody were added to a constant concentration of GGPD-L in the assay mixture and the rate of the resultant inhibition of GGPD activity measured. The method is direct, without any intermediate separation step, and gives a 50% decrease from the initial enzyme activity over the linear range. In an effort to extend the linearity to lower antibody concentrations, the experimental technique was altered by increasing the initial enzyme activity (GGPD-L) in order to obtain higher rates. No significant inhibition was observed under these conditions: blank (no anti-lidocaine), 5.62 nA/ min; 0.001 pg/mL anti-lidocaine, 5.48 nA/min. Therefore, to enhance inhibition, the antibody was premixed with the enzyme-antigen conjugate. The assay with 0.001 pg/mL anti-lidocaine was repeated with 5- and 10-min incubations. The respective rates were 5.10 and 4.96 nA/min. To limit assay time while still obtaining sufficient inhibition, a 5-min incubation time was selected as part of the experimental protocol for low antibody samples. The resulting calibration curve (average of two measurements per point, each pair less than 3% RAD), showed linearity between 0.001 and 0.01 pg/mL with a 25% loss in enzyme activity over this range. The slope in this case was -126, and r2 equaled 0.9852. While this second procedure is slightly more complicated, it also has a better sensitivity than the first. Inspection of Figure 4 reveals an order of magnitude shift in the linear range with the different experimental procedures. In effect, then, the higher enzyme (premix experiments) with its concomitant increase in antigen served only to shift the linear portion of the calibration curve rather than extend it. This shift is not attributed to the difference in enzyme concentration. The determination of KM for unconjugated GGPD at 0.1 unit and 0.02 unit does not indicate a change in enzyme kinetics. The KM values obtained from double reciprocal plots were 0.186 mM and 0.181 mM for the high and low GGPD, respectively. Because of the variation in the number of lidocaines bound to the enzyme (ZO),GGPD can act as a multiple-site antigen and is likely to undergo both inter- and intramolecular cross-linking during the immunochemical reaction (21). The extent and effect of these competing processes on the sensitivity of the technique described here are not easily discerned.
Anal. Chem. 1086, 58, 1245-1248
The order of magnitude shift of the calibration curves may be due primarily to the well-recognized phenomenon of required antibody-antigen equivalency, which depends upon the initial concentration of both reactants (22). The precision of the method was determined with and without antibody. The blank assay was repeated 8 times over an 8-h period giving 5.1% relative standard 'deviation (RSD). Eight measurements of the antibody assay, containing 0.083 pg/mL anti-lidocaine, were taken over a 4-h period yielding a 4.9% RSD. Platinum is susceptible to poisoning by oxidation and contamination from the oxidation products of NADH. Therefore, the absolute values of the rates were checked for indications of a change in the Pt response with time. No trend of increasing or decreasing response was evident. As a further point of reference a standard HzOz solution was tested during the latter precision study. The HzOz determinations, 11 points, had a 4.1% RSD. The electrochemical determination of anti-lidocaine as discussed here provides an example for rapid, separation-free measurement of antibodies. The coefficient of determination of both regression lines (significant at 99% confidence level) gives merit to the described methods for high and low antibody concentrations. In its simplest form, without preincubation, the assay can be used as a qualitative procedure. With stricter adherence to possible interferences and appropriate care of the platinum probe, the technique offers quantitative measurement of antibodies over restricted concentration ranges at nanogram levels. Registry No. GGPD, 9001-40-5; lidocaine, 137-58-6.
1245
LITERATURE CITED (1) Langone, J. L.; Van Vunakis, H. Methods Enzymol. 1983, 9 2 . (2) Schall, R. F.; Tenoso, H. J. Clin. Chem. (Winston-Salem, N . C . )1981, 2 7 , 1157-1164. (3) Van Vunakis, H.; Langone, J. L. Methods Enzymol. 1980, 70. (4) Crowle, A. J. Adv. Clin. Chem. 1978, 2 0 , 181-224. (5) Blake, C.; Gould, B. J. Analyst (London) 1984, 109, 533-547. (6) Monroe, D. Anal. Chem. 1984, 56, 921A-931A. (7) Rubenstein, K. E. Scand. J . Immunol. Suppl. 7 1978, 8 , 57-62. (8) Shiba, K.; Umezawa, Y.; Watanabe, T.; Ogawa, S.; Fujiwara, S. Anal. Chem. 1982, 5 2 , 1610-1613. (9) D'Orazio, P.; Rechnltz, G. A. Anal. Chlm. Acta 1979, fO9, 25-31. (10) Soisky, R. L.; Rechnitz, G. A. Anal. Chim. Acta 1978, 9 9 , 241-246. (11) Aizawa, M.; Suzuki, S.; Nagamura, Y.; Shinohara, R.; Ishlguro, I. Chem. Lett. 1977, 7 , 779-782. (12) Janata, J. J. Am. Chem. SOC.1975, 9 7 , 2914-2916. (13) Brontman, S. B.; Meyerhoff, M. E. Anal. Chim. Acta 1984, 162, 363-367. (14) Ngo, T. T.; Lenhoff, H. M. Appl. Blochem. Blotechnol. 1981, 6 , 53-64. (15) Finiey, P. R.; Williams, R. J.; Lichti, D. A. Clin. Chem. (Winston-&/em, N . C . ) 1980, 2 6 , 1723-1726. (18) Wehmeyer, K. R.; Doyle, M. J.; Wright, D. S.; Eggers, H. M.; Halsali, H. B.; Heineman, W. R. J . Lip. Chromatogr. 1983, 6 , 2141-2156. (17) Eggers, H. M.; Haisail, H. B.; Heineman, W. R. Clin. Chem. (WinstonSalem, N . C . ) 1982, 2 8 , 1848-1851. (18) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 47, 1337-1343. (19) Cunningham, J. A.; Underwood, A. L. Arch. Biochem. Biophys. 1966, 117, 88-92. (20) Singh, P. U S . Patent 4069 105, Jan 17, 1978 (Syva Co.). (21) Hohman, R. J.; Rhee, S. G.; Stadtman, E. R. R o c . Nafl. Acad. Scl. USA 1980, 7 7 , 7410-7414. (22) Myrvik, Q. L.; Weiser, R. S. Fundamentals of Immunology, 2nd ed.; Lea and Febiger: Philadelphia, PA, 1984.
RECEIVED for review August 27,1985. Resubmitted December 23, 1985. Accepted January 23, 1986. We gratefully acknowledge the financial support of NIH Grant GM-25308.
CORRESPONDENCE ~~
~~
Quenched Peroxyoxalate Chemiluminescence as a New Detection Principle in Flow Injection Analysis and Liquid Chromatography Sir: Peroxyoxalate chemiluminescence is known as a method for the analysis of HzOz (1-4)and certain fluorophores (5-8). Thus far we have confined our attention to the development of a hydrogen peroxide monitor based on the use of solid bis(2,4,6-trichlorophenyl)oxalate (TCPO) and an immobilized fluorophore. Recently, we found that certain easily oxidizable compounds such as sulfite, nitrite, anilines, and organosulfur compounds quench peroxyoxalate chemiluminescence. This phenomenon is of importance for at least two reasons. First, it gives an insight in the role of possible interferences on analytical procedures using the peroxyoxalate reaction. Second, a prelimifiary survey shows that under favorable conditions (relatively high H202 concentration and immobilized fluorophore) the analytical potential of peroxyoxalate chemiluminescence can be extended to the analyses of quenchers. Equations 1-4 presumably apply for the reaction of TCPO, HzOz, and fluorophore (F) in the presence of a quencher (Q); TCP is 2,4,6-trichlorophenol. This mechanism, apart from the quenching step Za, was proposed by Rauhut et al. (9) and modified by McCapra (10) introducing the electron transfer steps 2 and 3. The type of mechanism, known as the chemically induced electron exchange (CIEEL) mechanism, has extensively been studied by Schuster et al. (11,I.Z). Nevertheless uncertainty still prevails not in the least due to the
I
-0
L
-0
-0
I
1
products
3
*
F
--+
* F
F+hu
4
F+heat
4a
difficulties in positively identifying the intermediate C204(13, 14). All these studies, however, indicate that electron transfer
0003-2700/86/0358-1245$01.50/00 1986 American Chemical Society
