68
Anal. Chem. 1982, 54, 68-71
analysis of a CD active drug only if they are present in excess and absorb in the UV spectral range, for the reason given above. It is conceivable that these achiral molecules can be quantitated if an extrinsic CD signal is induced (15). The remaining interference in this case is that other additives might also be susceptible to an induced CD signal, but complications would only rise if that ingredient absorbed radiant energy in the W range of the drug of interest, namely, heroin in this case. Under these circumstances separation might be necessary. A distinct advantage CD has over GC or mass spectrometry is the much shorter time needed to complete the quantitative determination. Separation and derivatization are not required and the instrument does not have to be calibrated once the molar ellipticity coefficient is known. The recommended procedure for heroin analysis would be to take the anonymous sample into a pH 8.6 buffer solution to first qualitatively identify the compound and then measure the ellipticity at 253 nm after the addition of sodium hydroxide. The actual time for quantitative analysis is 20 min, which includes weighing and centrifugation. CD spectropolarimetry is therefore a strong competitor as an analytical method for the quick and reliable determination of heroin in solid specimens.
and the Oklahoma State Bureau of Investigation for providing the confiscates. We also thank H. L. Gearhart for the use of the GC equipment.
ACKNOWLEDGMENT
RECEIVED for review July 17,1981. Accepted October 13,1981. This work was supported by the National Science Foundation NSF-CHE-7909388.
We thank the National Institute for Drug Abuse and the Research Triangle Institute for providing the standard samples
LITERATURE CITED (1) Clarke, E. G. C. “Isolation and Identlficatlon of Drugs”; The Pharmaceutical Press: London, 1978;Vol. 1. (2) FuAon, C. C. “Modern Microcrystal Tests for Drugs”; Interscience: New York, 1969. (3) Manura, J. J.; Chao, J. M.; Safersteln, R. J . Forensic Scl. 1978, 23, 44-56. (4) DeZan, P.; Fasanello, J. J . Chromatogr. Sci. 1872, 70, 333-335. (5) Moffatt, A. C. J . Chromatogr. Rev. 1075, 773,69-95. (6) Clark, A. B.; Miller, M. D. J . Forensic Scl. 1978, 23, 21-28. (7) Siek, T. J.; Oslewlcz, R. J.; Bath, R. J. J . Forenslc Sol. 1975, 20, 18-30. (8) Nakamura, G.R.; Noguchl, T. T.; Jackson, D.; Banks, D. Anal. Chem. 1972, 4 4 , 408-410. (9) Chao, J. M.; Safersteln, R.; Manura, J. J. Anal. Chem. 1974, 46, 296-298. (IO) Bowen, J. M.; Purdie, N. Anal. Chem. 1980, 52, 573-575. (11)Bowen, J. M.; Crone, T. A.; Hermann, A. 0.;Purdie, N. Anal. Chem. 1980, 52, 2436-2440. (12) Crone, T. A.; Purdle, N. Anal. Chem. 1981, 53, 17-21. (13) Bowen, J. M.; Crone, T. A.; Head, V. L.; McMorrow, H. A.; Kennedy,
R. K.; Purdie, N. J . Forensic Scl., in press. (14) Bowen, J. M.;Purdie, N. J . Pharm. Scl., in press. (15) Bowen, J. M.; Purdie, N. Anal. Chem., in press.
Enzyme-Linked Immunoassay of Human Immunoglobulin G with the Fluoride Ion Selective Electrode Peter W. Alexander* and Carmellta Maltra School of Chemlstty, Universl(y of New South Wales, P.O.
Box 1, Kensington,
A fluoride-selective electrode has been used to detect Immunoglobulin G (IgG) In human serum to a level of 0.9 pg/mL. The method Is based on the selective detection of the horseradish peroxldase (HRP) label on the antibody after dlssolvlng the precipkate resuklng from the lmmunoreaction between IgG and HRP-labeled anti-IgG. The HRP label is quantkated by detecting the fluoride Ion released as a result of the HRP-catalyzed oxidation of p-fiuoroanliine by hydrogen peroxide. The fluoride ion concentration Is shown to be proportlonal to the IgG concentration precipitated In the region of antibody excess, without Interference from serum matrix components.
During the last 7 years applications of ion-selective electrodes (ISE) have been extended to include immunoassays. Methods of analysis of various immunoagents have been described in the literature as more new electrodes selective to organic compounds are developed and new uses for conventional electrodes are found for direct detection of the immunoagent (1-5) or of an appropriate label (6-10). Thus, the AgzS membrane electrode has been used in the immunoassay of human serum albumin in a semiautomated system ( I ) and the anti-benzoate antibody in a fully automated system (2).
New South Wales, Australia 2033
A number of other electrode immunoassay methods are now available. Hepatitis-B surface antigen (Hb,Ag) was detected with an iodide membrane electrode using HRP-labeled anti-Hb,Ag (3). An ammonia electrode was used (5) for the detection of bovine serum albumin (BSA) and cyclic adenosine-3’,5’-monophosphoricacid (c-AMP) via detection of the urease label after separation with the double antibody solid phase (DASP) technique described previously by Van Weeman et al. ( 4 ) . A trimethylphenylammonium cation (TMPA+) electrode (6) was used to detect the hemolysin antibody or complement which can cause the specific lysis of TMPA+ loaded erythrocytes (7) by detection of the TMPA+. Novel specific “immunoelectrodes” have also been developed, e.g., the Concanavalin A (Con A) immunoelectrode selective to yeast mannan (8) and the anti-human chorionic gonadotropin (anti-HCG) immunoelectrode selective to HCG (9, 10). With the exception of the use of TMPA+ marked erythrocytes for the detection of the hemolysin antibody or complement, all of the methods so far developed for immunoassay by ISE are heterogeneous. In contrast, the voltammetric immunoassays developed (11, 12) were based on the homogeneous competitive binding technique. Weber and Purdy (11)applied the technique to a model system involving codeine and morphine where
0003-2700/82/0354-0088$0 1.25/0 0 1961 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982 6Q
Flgure 1. A schematic diagram of the flow system for HRP detection: (a) indicator electrode, (b) reifarence electrode, (c) flow-through cell, (d) proportioning pump, (e) rni\//pH meter, (f) recorder, (g,) first mixing coil, (en)second mixing coil, (h) debubbler, (i)buffer solution, (I) clrcuiating water from constant temperature bath. The numbers in parentheses refer to the flow rates (mL/min) iri each flow channel.
morphine acted as an elect roactive antigen and codeine as the electroinactive antigen competing for antibody binding sites. Competitive binding between the two molecules resulted in release of morphine from the antibody complex with a subsequent increase in current signal due to unbound morphine. Heineman et al. (12) used a similar approach to determine estriol with the use of &-labeled estriol. Estriol and Hglabeled estriol compete for the limited binding site on the estriol antibody. An amperometric technique has been used by Aizawa et al. (13) who determined immunoglobulin G (IgG) by a competitive binding technique involving catalase labeled IgG and IgG. A Clark-type oxygen electrode was used. Yuan et al. (14) also reported an amperometric technique with a platinum electrode for immunoeledmchemical determination of creatine kinase isoenzyme MB. We now report an enzyme-linked immunoassay method for IgG based on the precipitin (15) technique using a fluorideselective electrode and HliP-labeled anti-IgG. In this method, the complete shape of the precipitin reaction curve is determined by specifically precipitating various amounts of human IgG with anti-human IgGI labeled with HRP, and the enzyme activity of the washed precipitates is determined by analysis of the washed precipitates in a continuous-flow electrode system after dissolution of the precipitin in dilute acetate buffer. The analysis is based on the continuous-flow detection of the liberated fluoride after the enzymatic reaction between the HRP-H202 complex and p-fluoroaniline resulting in the cleavage of a C-F bond. The concentration of the liberated fluoride is shown to be proportional to the H R P activity. Studies are reported showing the design of the flow system required for determination of the fluoride product of the enzyme reaction, the reslponse of the electrode to changes in enzyme concentration, amd quantitative calibration for de. termination of the enzyme-labeled antibody and of the antigen-antibody reaction. The sensitivity of the method it1 shown to be sufficient to detect IgG in tlhe fig/mL concen. tration range with negligible blank readings for unlabeled1 antiserum taken througlh the same procedure. EXPERIMENTAL SECTION Instrumentation. The continuous-flow system for HRP detection is shown in Figuire 1. Potentiometric measurements were done with an Orion Model 701A pH/mV meter coupled tot a Mace FBQ 100 strip chart recorder. The electrodes used were an Orion fluoride-selectiveelectrode, Model 94-09, provided with a flow-through cap of construction previously described (16) andl an Orion double junction wference electrode, Model 90-02, with 10% KN09 (w/v) solution in the outer compartment. The constant-temperature bath wm set at 36 "C to obtain more sensitive
response at low concentrations of HRP. Reagents. The reagents used in the flow system were 4.45 X M HzOzas substrate; 0.104 M p-fluoroaniline (dissolved in 0.16 M NaOAc-HOAc buffer, pH 4.7) as hydrogen donor, and horseradish peroxidase, type 11, of specific activity, 100 units/mg, lot no. 1259124, from Boehringer Mannheim, Sydney. The HRP-rabbft anti-human IgG (H and L chain) conjugate, lot no. S589, code no. 61-231,prepared according to the coupling technique described by Avrameas (17), was purchased from Miles-Yeda Ltd., Israel, with labeled enzyme activity of 520 units/mL. The human IgG standard was from Behringwerke, Germany, batch number A 375H, and of potency 193 mg/100 mL (1ampule with 81.47 mg of lyophilized serum contains 100 I.U. each of IgG, IgA, and IgM). The rabbit anti-human IgG was obtained from ORCM, Behringwerke, Germany, K- / lot no. 03106, 1.7 mg/mL (equivalent to 19 I.U./mL). The saline (0.17% NaC1, w/v) solution was prepared from analytical reagent grade chemical. Standard HRP solutions were prepared by taking 20,50,100, 200, 400, 600, 800, 1000, and 2000 pL of a stock HRP solution of concentration 27.6 units/mL and diluting to 100.0 mL. The labeled antibody solution was standardized against these HRP solutions. To test the enzyme activity of the labeled antiserum, we diluted 100 pL of the labeled antibody solution to 10.0 mL, and volum s ranging from 20 to lo00 pL of this solution were then diluted to 0.0 mL. Each sample was consecutively pumped into the flow system shown in Figure 1 with a fixed amount of the substrate and the donor in buffer. The concentration of fluoride produced by the enzymatic reaction was monitored with a fluoride electrode. The enzyme concentrations were then read off the curve. A precipitin curve for the reaction was constructed in the following way. Increasing volumes (3-50 fiL) of the standard IgG serum sample containing 193 mg/100 mL were added to a fixed quantity (100 pL) of the undiluted conjugate of potency in the range 1-2 mg/mL,. The total volume was kept constant in thecentrifuge tubes by adding distilled water to give 0.15 mL total volume. The precipitates were incubated for 1 h at 37 "C and left overnight in a refrigerator at 4 "C. The tubes were then centrifuged for 20 min and the supernatant was drawn out and transferred to a clean sample tube. The precipitates were washed twice with 1 mL of cold saline solution and once with 1 mL of cold water. After the final washing and decanting, the precipitates were dissolved in 1.0 mL of 0.16 M NaOAc-HOAc buffer. The solutions were then quantitatively transferred to a 5.0-mL volumetric flask and diluted to volume with distilled water. The HRP activity of the solution was determined by using a continuous-flow system, shown in Figure 1,by aspiration into the continuously flowing stream, using reagent concentrations given above. The sampling rate was 12 per hour, with a sample-to-wash ratio of 1:8, using in this case a 0.03 M NaOAc-HOAc buffer as wash, instead of water. Peak heights for each of the test solutions were recorded and plotted as a Ifunction of antigen concentration. The Supernatant was analyzed by taking an aliquot of 30 fiL and diluting to 25.0 mL total volume with distilled water. This was also analyzed in the continuous-flow system to determine enzyme activity as described above. As a blank, the precipitate, obtained by adding 100 pL of the unlabeled antiserum of concentration 1.7 mg/mL to 20 pL of the IgG standard solution (193 mg/100 mL), was washed and dissolved in 1mL of 0.16 M NaOAc-HOAc buffer and diluted to 5.0 mL.
f
RESULTS This study wm aimed at establishing that the HRP enzyme label on an IgG antibody can be detected with the fluoride electrode. By use of immuno-precipitin technique to specifically precipitate IgG from serum samples, the enzymelabeled antibody was specifically precipitated and serum matrix components were then washed out according to established methods (15). The HRP activities of both the whole enzyme labeled antiserum and the labeled precipitate were then determined in a continuous-flow system shown in Figure 1, using an aqueous H R P solution as a standard.
70
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982 I
Table I. Response Time of the Orion Fluoride Electrode under Flow Conditions
I_
concn of fluoride, M 1x 1x 1x 1x 1x
tws
10-6 10-5 10-4 10-3 10-2
a
b
58
138
80 26
298 269
10 5
355 473
a When fluoride concentration increases from nil to the indicated value. When fluoride concentration decreases from the indicated value to nil.
h
48
-0
24
36
h
h
h
h
h
12
Sampling Time i m n l
Flgure 2.
Sample peaks corresponding to various activities of aqueous
HRP standards: (a) 5.5 X IO3, (b) 1.4 X lo-*, (c)2.8 X lo-*, (d) 5.5 X (e) 1.1 X IO-', (f) 1.7 X IO-', (9)2.2 X lo-', (h) 2.8 X IO-', (i) 5.5 x IO-' unit/mL. 168
144
120
72
96 Sampling Time
48
24
0
lmlnl
Flgure 4. Sample peaks corresponding to various activities of HRPconJugatedantl-human IgG: (a) 2.0 X (b) 7.5 X (c) 1.5 X lo-', (d) 2.1 X IO-', (e) 0.05,(f) 0.08, (9) 0.10, (h) 0.12 units/mL.
04iO'
'
'
'
'
-20
Log
HRP
'
'
' -1' 0 UnllS/mL
'
'
ACtlYltV
'
'
'
b'
Flgure 3. Calibration plot of peak height (mV) against HRP activity (units/mL) for aqueous HRP standards.
Determination of HRP Activity. The enzyme reaction used in this study was the HRP-catalyzed oxidation of p fluoroaniline by hydrogen peroxide. The overall reaction can be written according to that proposed by Hughes and Saunders (18) as 2 H
p
O
-
- 1232
r
o
N
e
\
H
+
h'*
F
+
2H2O
Optimized reaction conditions for determination of the released fluoride ion with the selective electrode were determined initially in this laboratory (19) and will be published in detail a t a later date. However, we found that buffering the donor p-fluoroaniline solution a t pH 4.7 gave optimum sensitivity for determintion of H R P activity. The fluoride electrode was then used in the continuous-flow system shown in Figure 1with the optimized donor and substrate concentrations, temperature, and buffer pH given in the Experimental Section. Figure 2 shows the sample peaks recorded when aqueous H R P standards were fed into the flow system, and the corresonding calibration plot for HRP activity is shown in Figure 3. The enzyme was detectable in the activity range from approximately 0.005 to 0.80 units/mL. Electrode Response Time. The results in Figure 2 indicate a slow electrode response to changes in sample enzyme activity, particularly during the wash cycle. Table I shows
the response time of the fluoride electrode in this flow system when fluoride standards were fed through the system in place of enzyme solutions. Because of the slow response of up to 8 min for a change from high to low fluoride concentration, the sampling was operated under non-steady-state conditions with a short sampling time of 30 s and a long wash between samples of 4 min. The sampling rate used for determination of enzyme activity was therefore limited to approximately 12 samples per hour, as shown in Figure 2. The slow electrode response in this system is due to the high concentration of donor in the stream, since the response is also very slow when fluoride is added to the stream in the absence of protein. Labeled Antiserum Samples. The IgG antiserum labeled with HRP was also tested for enzyme activity in the same flow system and was standardized against the aqueous HRP standards. After dilution of the commercially supplied antiserum, samples fed through the flow system gave similar peaks to the aqueous H R P standards, as shown in Figure 4, without any obvious interference from the serum matrix components. The peak heights in Figure 4 were used to determine the enzyme activity of the diluted antiserum solutions from the calibration plot in Figure 3, giving enzyme activities in the range 0.002-0.12 units/mL. Labeled Antibody Precipitation. For determination of human IgG in serum samples, the IgG was precipitated by mixing with a labeled antibody solution. The precipitate was collected and washed to remove unprecipitated antibody and, after redissolving, was fed through the flow system, as before, to determine enzyme activity. The sample peaks recorded gave the same response times as shown in Figure 4 for whole antiserum samples. A calibration plot is shown in Figure 5 resulting from the reaction of increasing quantities of IgG with a fixed amount of the labeled antibody. The curve showed the typical shape of a so-called precipitin curve (15) with three distinct precipitin zones: (A) the zone of antibody excess, (B) the equivalent zone in which neither antigent nor antibody can be detected in the supernatant, and (C) the zone of antigen excess where the
ANALYTICAL CHE-MISTRY, VOL. 54, NO. 1, JANUARY 1982
O
L
d 2
o'.T
0'6
L o g 1gG Conc
lb
0;
12
14
M I m L
Figure 5. Precipitin curve for the IgG-anti-human I g G (HRP-labeled) immunoreaction showing tho three precipitin zones: (A) antibody excess; (B) equivalence; (C) antigen excess.
Table 11. Precision of the Individual Meiasurements for Precipitin Curve Construction IgG concn, fig/mL 1.16 1.54 1.93 3.09 7.72 12.74 a
meana peak height, m V
std dev
'14.9 122.3 154.8 139.3
0.2 0.2 0.4
107.1
2.0 0.9
'73.5
1.0
% RSD 2.0
1.0 0.7 2.0 2.0
1.0
Mean of four replicates.
precipitate redissolves. The precision of replicate pea:k measurements for each of the IgG samples analyzed is shown in Table 11,giving % RSD values within about 2% for fluoride electrode determinations at a sampling rate of 12 per hour. The observed results indicate that the electrode method is capable of detecting changes in the antibody-antigen ratio over the entire course of the reaction in agreement witlh previous precipitin curve studies (15)and that the HRP en.zyme label retains its ;activity after immunoprecipitation. The calibration curve .inFigure 5 shows .that valid analytical results are obtained only in the region of antibody excess, in agreement with classical .techniques (20). Providing this 1im.itation is recognized, we have shown that the fluoride electrode method is applicable to the analysis of serum samples by use of serum standards, not aqueous IgG standards. The IgG serum standards we used are supplied commercially witlh certified IgG content obtained by clamical precipitation methods. Possible interferences present in the serum samples are removed during washing of the precipitate. Direct analysis of the supernatant should be possible with the fluoride electrode technique, but only if the antiserum used contains HRP conjugates with intact antigenic properties. For the blank read&, unlabeled antibody-antigen precipitin was treated in the same manner as the labeled antibody-antigen precipitin and passed through the automated system. A negligible blank (
