2355
Anal. Chem. lQ84, 56, 2355-2360
Enzyme-Linked Immunoadsorbent Assay with Electrochemical Detection for a,-Acid Glycoprotein Matthew J. Doyle,’ H. Brian Halsall,* and William R. Heineman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172
An enzyme-linked lmmunoadsorbent voltammetrlc assay based on the detection of catalytically generated electroactlve product has been Investlgated. Human orosomucold (OMD, a,-acid glycoprotein) was cross-llnked to alkaline phosphatase (AP, EC 3.1.3.1) uslng glutaraldehyde. The physlcal, chemical, and lmmunologlcal properties of the OMD-AP conjugate compared favorably with those of the natlve precursors. AP-generated phenol was detected amperometrlcally at a carbon paste electrode by llquld chromatography/electrochemlstry (LCEC). The detectlon limlt for OMD Is 1.0 WmL, and the assay Is most senSnlve between 1.0 and 10.0 ng/mL. The LCEC method exhiblts a 3-5% relatlve standard devlatlon.
Immunoassay is a very selective and sensitive approach to trace metabolite analysis (I).The interaction between antigen (Ag) and antibody (Ab) molecules may be extremely specific under favorable conditions as a consequence of binding geometries (2). The utility of immunoassay in diagnostic medicine is well documented, and a number of routine clinical methods have been established for the determination of a wide variety of common antigens (3). One form of immunoassay is the competitive binding or saturation analysis assay (4). A competitive equilibrium is established between excess labeled (Ag*) and unlabeled antigen (Ag) for a limited amount of highly specific Ab as outlined in Scheme I. Following saturation of the Ab binding sites, the relative amounts of “bound” and “free” label are determined and a standard curve plotted. As a result of mass action, the bound-to-free ratio (B/F) of any test solution is proportional to the concentration of Ag originally present in that solution. In this manner, blood and urine samples are routinely screened for relative levels of proteins, steroids, hormones, and drugs. Heterogeneous techniques require physical separation of bound and free Ag* prior to distribution analysis. Since the pioneering work of Yalow and Berson (5,6) in 1959, radioisotopeshave been employed as the label of choice. However, a number of nonisotopic alternatives have been developed including electron spin resonance for detecting radical labels (7)) nephelometry (8), fluorescence (91,chemiluminescence (lo),and enzyme labels (11)among others (12-14). The wide dynamic range and low detection limits of modern electroanalytical methods (15)make labeling the analyte with an electroactive label an attractive approach to immunoassay technology. In addition, electrochemical procedures are convenient, fast, inexpensive, and selective. Both potentiometric and voltammetric approaches to electrochemically based assays have been investigated. The former employs ion-selectiveor chemically modified electrodes as the potentiometric sensors. Yamamoto et al. (16)linked Current address: The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 39175, Cincinnati, OH 45247. 0003-2700/84/0358-2355$01.50/0
Scheme I. Competitive Binding Immunoassay A g +
As”
/
Ag :Ab
Ag”: Ab
antisera specific for choriogonadotropin to a titanium wire. Reaction with Ag resulted in a positive shift in electrode potential vs. a urea reference electrode. Alexander and Maltra (17) utilized a fluoride electrode to monitor horse radish peroxidase activity during an IgG immunoassay. Meyerhoff and Rechnitz (18)and Gebauer and Rechnitz (19) applied an ammonia gas-sensing electrode in assays for urease-labeled bovine serum albumin (BSA),cyclic adenosine monophosphate (CAMP), and adenosine deaminase-labeled dinitrophenyl (DNP), respectively. In addition, Rechnitz and co-workers (20,21) have constructed (trimethylpheny1)ammonium (TMPA+) electrodes and used them as detectors in TMPA’ and complement mediated immunoassays. A review of the use of immunospecific electrodes including the applications of field effect transistors can be found elsewhere (22). The finite current techniques of amperometry and voltammetry comprise the second class of electrochemically based immunoassays. Weber and Purdy (23,24)developed a continuous flow, homogeneous voltammetric immunoassay (VIA) for the determination of morphine in the presence of codeine at a glassy carbon electrode. Breyer and Radcliff (25)and Zikan and Kotynek (26)assessed the interactions between azo-labeled protein, nitrophenyl-labeled lysine, and their specific antisera. Wehmeyer et al. (27)have reported the development of a homogeneous VIA for estriol labeled in the 2- and 4-positions with nitro groups using differential pulse polarographic detection. Binding of 2,4-dinitroestriol to specific antisera resulted in a decrease in the corresponding reduction current. Aizawa and co-workershave developed a series of assays for IgG (28),choriogonadotropin (29))and a-fetoprotein (30)based upon electrodes covered with membrane bound antibodies. Native Ag and enzyme-labeledAg* were allowed to equilibrate a t the surface, and the enzyme product was determined amperometrically. Additionally, Alam and Christian (31)have reported a VIA for human serum albumin (HSA) in which lead bound nonspecificallyto HSA serves as an indicator. We have described a heterogeneous VIA for proteins based upon a metal chelate label (32-34), using indium as the indicator metal. The indium-labeled protein complex was stable until the metal was purposefully released by acidification for determination by anodic stripping voltammetry. The sensitivity and detection limits of electrochemical approaches are enhanced when coupled to chemical amplification mechanisms such as enzyme catalysis (35).The activities of a number of enzymes have been monitored amperometrically (36-38))and enzyme immunoassay in some cases has been shown to have the same intrinsic sensitivity as RIA (39,40).Yuan et al. (41)have described a homoge0 1984 Amerlcan Chemlcal Soclety
2358
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Scheme 11. AP Hydrolysis of Phenyl Phosphate
electrochemical detection (LCEC).
H
0
0
H
phenol
phenyl phosphate
neous enzyme VIA for creatine kinase isoenzyme MI3 by using a Pt electrode and coupling NADH to the ferricyanide-diaphorase indicator reaction. Eggers et al. (42)employed flow injection analysis with an enzyme-coupled VIA for the determination of phenytoin using the EMIT system. The rationale for using enzyme labels extends beyond sensitivity considerations. Some enzymes are highly selective for a specific substrate, and rate methods can be employed to minimize error due to competing side reactions. Many are commercially available in high purity and at low cost. Several enzymes are readily adaptable to electrochemical methods and generate electroactive product from an electroinactive, and therefore noninterfering, substrate. Alkaline phosphatase (EC 3.1.3.1, AP) catalyzes the hydrolysis of phosphate esters (43) as depicted in Scheme 11. Phenol can be oxidized (El,2= +750 mV vs. Ag/AgCl in 0.05 M carbonate buffer) whereas the substrate phenyl phosphate is electroinactive a t positive potentials. An attractive feature of this system is the evolution of an oxidizable product, which eliminates the need to deoxygenate the sample and mobile phase prior to analysis as is required in reductive electrochemical experiments. Electrochemical methods for the assay of AP (44) and the determination of phenol have been described (45). The antigen that was chosen for these model studies was human serum orosomucoid (OMD, q-acid glycoprotein). OMD is a small glycoprotein (41 000 daltons) comprised of 40% carbohydrate by weight, 10-12% of which is sialic acid (46). OMD is produced in hepatic tissue and consists of a single polypeptide chain containing two disulfides. It is an acute p h e reactant during inflammation (47),pregnancy (481, and cancer (49). Serum OMD levels have been shown to vary dramatically during the course of certain malignancies (50, 51). Altered OMD-drug binding affinities correlate directly with metastatic progression (52,53),and the diagnostic value of OMD as a tumoral marker has been suggested (54). Despite knowledge of its structure and interactions (55-57), the physiologic role of OMD has still not been defined. Quantitative methods for the determination of OMD are quite tedious and often insensitive. Common procedures include seromucoid precipitation (58),radial immunodiffusion (59), electrophoresis (60), immunoelectrophoresis (61),enzyme enhancement assays (62),and more recently ELISA (63). The development of a sensitive, simple, and inexpensive OMD assay may result in a useful screen for several pathological conditions and aid in the elucidation of its physiologic function. Protein-protein coupling (OMD to the AP label) can be accomplished in a one-step procedure using a homobifunctional reagent such as glutaraldehyde (GA). GA-induced cross-links are irreversible and quite stable to acid hydrolysis (64). The reaction is simple, complete within 2 h, and is highly efficient. An attractive feature of the GA procedure is that it results in minimal loss of enzyme activity or antigen immunogenicity (65). These principles were applied to develop OMD-AP conjugates to be utilized as tracers in a solid-phase heterogeneous VIA. Evolution of enzymatically generated phenol was monitored by using liquid chromatography with thin layer
EXPERIMENTAL METHODS Apparatus. The liquid chromatograph was an LC-150 with a 20-pL sample injection loop and an LC-4B amperometric controller (Bioanalytical Systems, Inc., West Lafayette, IN). The analytical column and precolumn were 40 mm X 4.6 mm i.d. stainless steel (Universal Scientific, Atlanta, GA) and were slurry packed with 10 pm irregularly shaped RSiL (2-18 reverse-phase packing material (Alltech Associates, Deerfield, IL). The precolumn was placed between the pump and the injection valve to saturate the mobile phase while the analytical column separated phenol from the assay buffer. The TL-4A thin layer flow cell had a carbon paste-graphite-38-based working electrode (CP-oE), Ag/AgC1 reference electrode, and glassy carbon auxiliary electrode. A 5-mil gasket gave an 8.90-pL cell volume. Flow rates were typically between 0.4 and 0.6 mL/min. A 174A polarographic analyzer (Princeton Applied Research Corp., Princeton, NJ) was used for differential pulse voltammetry (DPV) at a carbon paste-carbon black electrode (CP-cbE). The CP-cbE was formulated from 3 g of carbon black (Cabot Corp., Boston, MA) in 12 mL of paraffin oil and packed into a 4 mm X 4 cm glass capillary tube with 2-mm i.d. A Pt wire was inserted in one end for electrical contact. Differential pulse voltammograms were recorded vs. SCE. A Polyanalyst disc gel apparatus (Buchler Institute, Fort Lee, NY) was used for electrophoretic studies. Gels were scanned at 570 nm by using a Gilford scanning spectrophotometer. UVvisible spectroscopic measurements were performed on a Cary 210 spectrophotometer (Varian Associates, Inc., Palo Alto, CA). Gel chromatography of the modified protein on Sephacryl-200 (Pharmacia, Piscataway, NJ) was controlled by a Minipuls 2 peristaltic pump (Gilson Electronics, Middleton, WI). An LKB 2138 Uvicord S UV flow monitor (LKB, Bromma, Sweden) set at 278 nm was the detector. Reagents and Solutions. All buffer solutions were prepared from distilled/deionized H20 of at least 106-Qresistivity using ACS grade chemicals obtained from MCB, Norwood, OH, or Fisher Scientific,Cincinnati, OH. The 0.15 M phosphate buffered saline (PBS, pH 7.4), 0.05 M carbonate (pH 9.6), and 0.08 M Tris/HCI (pH 8.0) buffers were prepared by using the appropriate sodium and ammonium salts, respectively. Tween 20 solutions (Fisher) were prepared as 0.5 mL of Tween 20 in 1L of 0.15 M PBS. Human OMD (Lot No. 81F-93610),bovine intestinal AP (Lot No. 811-8170), and 25% w/v GA were all obtained from Sigma Chemical, St. Louis, MO. OMD and AP migrated as single bands during electrophoresis and were used without further purification. Anti-OMD (Lot No. G33) and nonspecific IgG (Lot No. R566) were obtained from Miles-Yeda,Limited, Elkhart, IN. The mobile phase was a 0.10 M phosphate buffer (pH 7.0) that was filtered through 0.2-pm membrane filters (Gelman Sciences Inc., Ann Arbor, MI) and degassed by aspiration prior to use. Substrate solutions were always prepared just prior to use to prevent nonenzymatic hydrolysis. AP substrate solutions were prepared from 0.0146 g of phenyl phosphate (Calbiochem-Behring Corp., La Jolla, CA) and 0.0055 g of MgCI2.6H20(Baker, Phillipsburg, NJ) in 25 mL of 0.05 M carbonate, pH 9.6. All standard OMD and enzyme conjugate solutions were diluted by using PBS/Tween to minimize nonspecific interactions. Antibody coating solutions were prepared in 0.05 M carbonate buffer by adding the appropriate amount of antiserum. @-Mercaptoethanol, sodium dodecyl sulfate, and acrylamide reagents were all of electrophoretic grade (Bio-Rad, Richmond, CA). Protein Conjugation. The one-stepglutaraldehydeprocedure of Avrameas et al. (66) was employed as follows: alkaline phosphatase (AP, 4.55 mg in (NH4)2S04)was centrifuged for 100OOOg min and the (NH4)2S04aspirated away. The solid A P was dissolved in 2.0 mL of an OMD solution (0.239 mg/mL in PBS). The molar ratio of the resulting solution was 4.14/1 (AP/OMD). This mixture was dialyzed twice against 1L of PBS for 12 h. Then 0.40 mL of 1% w/v GA was slowly added, dropwise to the dialysate. The solution was vigorously shaken during the addition and allowed to stand at room temperature for 2 h. The resulting mixture was eluted on a Sephacryl-200column (1.05 cm x 54 cm) with Tris/HCI buffer (pH 8.0). Fractions were collected, HSA was added to a final concentration of 1mg/mL to stabilize
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 Competitive
L inlted
Add anti-OMD ( Y l t o polystyrene cuvette
2357
Enzyme
Irn rn unoadsor bent
Assay
Add excess Ag: sample (01, OMD - E lo1 I
0
-
120
60
1eo
Rt I m i n ) Determine [ p l
---+
Amperometrlcally By
\
DPV or LCEC
Chromatogram of the OMD-AP reaction mixture eluted with 0.08 M Tris/HCI (pH 8.0) on a Sephacryl-200 column (A = 280 nm). Fiow rate = 0.28 mLlmin. Flgure 2.
Add Substrate (SI
Figure 1.
A
Outline of the heterogeneous, solld-phase, enzyme-linked
VIA protocol 6 using poly(styrene) cuvettes.
the complex, and the conjugate was stored at 4 "C. Complex Characterization. Electrophoretic mobilities were measured in 10% poly(acrylamide)running gels and 3% stacking gels. Samples were treated with both /3-mercaptoethanol and sodium dodecyl sulfate for comparison. Ouchterlony radial immunodiffusion was performed as is customary. Warren assays were performed according to the method of O'Kennedy (67). Kinetic assessment of native and modified enzyme was carried out in classical Michaelis-Menten fashion. AP activity was assessed spectrophotometrically by monitoring p-nitrophenol evolution at 400 nm vs. time at specific substrate concentrations. VIA Protocol. The heterogeneous VIA-ELISA procedure for the determination of OMD is outlined in Figure 1. Specific Ab was passively sorbed on the walls of 10 mm X 5.5 mm poly(styrene) cuvettes (Gilford Instruments,Cleveland, OH). This solid-phase Ab matrix was then allowed to equilibrate with the test solution. Typically, 0.6 mL of OMD antiserum solution (1.0 pg/mL) or nonspecific IgG (10.0 pg/mL) was placed in each cuvette and incubated at 37 O C for 24 h. Each well was then aspirated dry and rinsed twice with PBS/Tween for 15 min and once rapidly. Rinsing with surfactant removes excess protein and minimizes nonspecific adsorption from occurringby coating unoccupied sites. Cuvettes can be conveniently stored at 4 "C or used directly. An OMD-containing solution, either an unknown or a standard (0.4 mL), was mixed with 0.1 mL of a 1/100 dilution of the enzyme conjugate (OMD-AP-I) and allowed to equilibrate with the Ab solid phase. Following a 12-h incubation period, the cuvettes were aspirated dry, again rinsed twice with PBS/Tween for 15 min, once with PBS/Tween quickly, and once using 0.05 M carbonate. The wells were aspirated dry, and 0.5 mL of substrate solution was added to each. Cuvettes were allowed to stand at room temperature for 1h, and the enzymatic reaction was stopped by transferring the substrate solutions from each well into corresponding poly(propy1ene) microbeakers. The product of the enzymatic reaction was determined amperometrically. LCEC samples were diluted as follows: 10 pL of each solution wm mixed with 1.0 mL of 0.05 M carbonate, and 20 pL of this diluted sample was injected into the chromatograph. Conversely, differential pulse voltammograms were obtained by using undiluted substrate solution. RESULTS AND DISCUSSION Characterization of t h e Enzyme Conjugate. Chromatographic analysis of the OMD-AP conjugate revealed a polydisperse synthetic mixture (Figure 2). Higher moleqular weight fractions were collected (i.e., OMD-AP-I) and employed in the final form of the assay. The higher molecular weight fractions are rich in the conjugate while lower molecular weight fractions contain unreacted OMD or OMD aggregates. Ouchterlony radial immunodiffusion gels were positive for both pooled fractions (I and 11) of OMD-AP. Thus, immu-
B ,Apt
HSA
0.16 a u I
OMD-A P - I
-0MD
OMD- AP-IT
Spectrophotometric scans at 570 nm of (A) OMD-AP chromatographic fractlons I and I1 and (B) OMD and AP separated on SDS poly(acrylam1de)electrophoretic gels. Flgure 3.
nogenicity is retained following the synthetic procedure. SDS electrophoretic gels calibrated for molecular weight were used to determine the molar ratio of AP to OMD in the conjugate. UV gel scans defined Rj bands for each macromolecule (Figure 3). OMD, HSA, and AP had Rf values of 0.83,0.68, and 0.63, respectively. Chromatographic fractions of OMD-AP reflected the heterogeneous nature of the reaction mixture. OMD-AP-Iand OMD-AP-I1 sample gels exhibited the presence of higher molecular weight components (Figure 3A). Severalbands (R, = 0.15,0.33) are HSA aggregates which were added as a stabilizing agent. Bands at Rj = 0.08 (AP,) and 0.41 (AP,) are polymers of AP itself while the band occurring at Rj = 0.23 represents the conjugate OMD-AP2. OMD-AP was not apparent in these scans. The bands at Rf = 0.83 (OMD) and 0.63 (AP + HSA) are due to unreacted constituents. This was expected since the glutaraldehyde linking reaction only approaches 60% efficiency (64). The integrity of the carbohydrate moiety of the modified OMD was evaluated via Warren assay (67). The assay is specific for 2-keto-3-deoxy sugar acids such as N-acetylneuraminic acid (NANA) and depends upon periodate-induced chromophore formation with thiobarbituric acid followed by cyclohexanone extraction. The amino group is usually substituted and hence unreactive. OMD generally contains between 10% and 12% NANA by weight. Hydrolysis (cleavage of NANA prior to chromophore formation) Warren assays indicated that a loss of reactive groups occurs following GA cross-linking (Table I). This may be caused by trapping of surface sugar residues between protein, cross-linking of sugars to primary amino acids, or modification of the sugar
2358
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. Warren Assay Data determined cNANA, pG % NANA
sample
conditions
OMD AP OMD-AP-I OMD-AP-I1 OMD OMD-OMD OMD-OMD
hydrolysis hydrolysis hydrolysis hydrolysis hydrolysis hydrolysis nonhvdrolvsis
5.17 0.74
0.8
c
OMD-AP CONDITION OPTIMIZATION
10.82
blank 0.00
0.00
trace
0.98 20.35 2.78 0.74
11.46 2.50
0.00
Table 11. Kinetic Parameters Comparison -15
Km,
method Michaelis-Menten LineweaverBurk Hanes-Woolf Woolf-Hofstee
plot v
vs. s
mM OMD-AP
pM/min AP OMD-AP
1.36
1.36
22.00
4.89
vs. s
1.49
1.10
25.70
6.35
vs. s vs. v / s
1.34 1.27
1.24 1.04
23.70 29.30
5.47 6.36
1.19 A 0.14
25.2 3.1
5.27 i 0.26
1/v
s/u v
AP
av values 1.37
*
0.09
-10
-05
00
05
10
15
Vmar:
moiety, preventing chromophore formation. GA-induced cleavage of sugars is precluded by the blank observation following the nonhydrolysis assay of self-coupled OMD. Despite apparent changes in associated carbohydrate, the OMD-AP complex retains immunoreactivity. This is not unexpected, since Krotoski and Weimer have reported that the glycan chains of OMD are poorly immunogenic (81). C k i d Michaelis-Menten kinetic analyses were performed to test the viability of the enzyme following modification by measuring KM and V-. AP also catalyzes the conversion of p-nitrophenyl phosphate (NPP) to p-nitrophenol (NP). The UV absorption spectra of both NPP and NP are sufficiently resolved to allow the evolution of NP to be monitored without interference at 400 nm. AP activity was monitored spectrophotometrically in a simple rate assay using the NPP substrate. These assays for AP and OMD-AP-I were performed at substrate concentrationsvarying between 0.1 mM and 10 mM. The average KM and V- values obtained are given in Table 11. There was little difference in average KM values between AP (1.37 mM) and OMD-AP-I (1.19 mM). Changes in Vwere apparent but are related to differences in enzyme concentration. These data agree well with published values (68). Thus, physical modification of AP upon coupling did not appreciably alter its activity or prevent substrate diffusion. LCEC/Voltammetric Evaluation. During developmental work phenol was detected by differential pulse voltammetry with the CP-cbE described in the Experimental Methods. The CP-cbE exhibited lower background currents and a higher conductivity than its graphite-38-based counterpart. The response of the CP-cbE toward phenol was linear between 30 and 400 ng/mL. A new electrode surface was generated for each analysis to minimize fouling phenomena. Such a practice greatly contributes to random error since a reproducible surface is difficult to achieve. Prudence dictated that emphasis be directed toward a method which exhibited increased electrode longevity. Hydrodynamic techniques such as LCEC are ideally suited when fouling is a problem since less than 5% of the electroactive species undergoes reaction a t the electrode surface. Since the immunoassay separation scheme results in relatively pure solutions of phenol and electroinactive substrate, phenol can be determined rapidly by simple flow injection
LOG Ab
COATING
CONC
Figure 4. Assay optlmizatlon matrix (I,, at +430 mV vs. SCE) for Ab coating concentratlon and enzyme conjugate dilution by differential pulse voltammetry at a CP-cbE electrode. analysis. However, the detection limit of this method is limited by the simultaneous occurrence of a void volume peak from the substrate buffer alone (45). Consequently, a (2-18 reversed-phase column was used to preferentially retain the phenol (ca. 3 min a t 0.5 mL/min). This procedure resulted in a linear standard curve for the determination of phenol over 2 orders of magnitude with a detection limit of 1.0 X M. A hydrodynamic voltammogram for phenol indicated that maximum sensitivity is obtained a t potentials greater than +850 mV vs. Ag/AgCl. Current attenuation resulting from electrode filming by adsorbed oxidation product was found to occur at concentrations greater than 6.0 X lo-’ M (45). Consequently the substrate reaction was stopped or diluted such that less than 1.0 X lo-’ M phenol would be injected on the column. Immunoassay Protocol. Since the “bound” and “free” labeled antigen are indistinguishable to substrate, a solid-phase heterogeneous assay scheme that included their separation was developed. IgG has been covalently linked to a number of support materials and employed as a solid phase for immunoassays. Materials such as silicone (69),glass (70),Teflon (711, and Sephadex (72) have been so modified. Catt and Tregear (73) have refined this technique to utilize poly(styrene) surfaces. The simpler and more reproducible method of hydrophobic adsorption has become favored and routinely utilized clinically (74, 75). The adsorption kinetics and stability of IgG molecules on a poly(styrene) matrix have been studied in detail (76).Poly(styrene) cuvettes were chosen as the support matrix for adsorbed OMD antiserum. Assay conditions were optimized by differential pulse voltammetry for the amount of OMD-AP conjugate added and the concentrationof the Ab coating solution. Best current response occurred a t an OMD-AP dilution of 1/100 and an Ab coating solution concentration of 1 pg/mL (Figure 4). Cuvettes coated with nonspecific IgG or Tween 20 exhibited no enzyme activity following incubation. Similarly, cuvettes coated with OMD antiserum (a-OMD), incubated with AP, and rinsed exhibited no enzyme activity. VIA Evaluation. Chromatograms for a series of OMD standards following VIA appear in Figure 5A-I. The concentration of phenol, and hence peak current, is inversely proportional to the concentration of OMD in the test solution. This relationship is as predicted when determining bound labeled analyte during a competitive saturation analysis assay. Nonspecific adsorption was not apparent under these test conditions (Figure 5J,K). A plot of peak current vs. OMD concentration indicated that the assay is most sensitive in the region between 1.0 and 10.0 ng/mL (Figure 6). Assay sensitivity can be tailored to cover a specific dynamic range within limits governing the amounts of Ab which can be sorbed to
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
0.1 nA
2359
variant forms of OMD are produced in some malignant states (80). Adaptation of the OMD assay described here for prognosis and diagnosis should result in a rapid, inexpensive, and automatable tool.
I
ACKNOWLEDGMENT H
h L min/cm
Flgure 5. V I A of a series of OMD standard solutions: (A) 0.05 M carbonate; (B) 200, (C) 100, (D) 60, (E) 10, (F) 5.0, (G) 2.5, (HI 1.0, and (I) 0.75 ng/mL. The addition of AP alQne (J) or OMD-AP (K) to cuvettes coated with nonspeclflc IgG.
90 80 70
60 2 W
a
50
2 V
40
20
$ 0
50 OM0
100
150
200
CONC. (ng/rnL)
Flgure 8. Typical standard curve (expanded scale insert) for the determination of a series of OMD solutions by VIA.
the solid phase. It is generally advisable to work at the lowest range possible, maximize assay sensitivity, and dilute the analyte to meet those conditions. The percent relative standard deviation for a 40 ng/mL OMD standard by LCEC was 3.7% (n = 9),much improved over the differential pulse voltammetry method (15-20% RSD). Rate methods can be employed to minimize error due to competing side reactions or quantitate analyte in the presence of interfering constituents. A plot of ips vs. substrate incubation time for a 5 ng/mL OMD standard following VIA was linear with a slope of 0.0228 nhlmin. The substrate incubation period may be shortened to 10-20 min without a loss in sensitivity, since a detectable amount of phenol was reproducibly generated in that time span. This is an extremely sensitive assay which should be readily adaptable to a number of macromolecular analytes of clinical importance. It offers the opportunity for taking patient samples as small as 0.4 ILLwhen the antigen concentration is only 1Mg/mL, distinctly advantageous in fetal and neonate procedures. Of particular interest are macromolecules whose presence or change in concentration is considered diagnostic for a particular pathology, especially cancer. Carcinoembryonic antigen(s) (CEA) have been a focus of interest in this regard for a number of years, and recent work (77) with high-affinity monoclonal antibodies to conformational determinants of CEA offers renewed hope for diagnostic specificity in its assay. Other recent reports (78,79) suggest that OMD and CEA are structurally related and further that
We acknowledgethe helpful comments of K. R. Wehmeyer, J. A. Wise, and M. L. Friedman. The assistance of Peter T. Kissinger, Bioanalflical Systems, Inc., and Lawrence Kaplan of the University of Cincinnati Medical Center is gratefully appreciated. The carbon black material utilized in these studies was the generous gift of Robert E. Doyle and the Cabot Corporation. Registry No. Phenol, 108-95-2.
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RECEIVED for review January 23,1984. Resubmitted June 13, 1984. Accepted June 13,1984. This work was supported by NIH Grants A116753 and HD13207, NSF Grant CHE8217045, and a summer ACS Analytical Division Research Fellowship sponsored by the ACS Division of Analytical Chemists (M.J.D.). M.J.D. also acknowledges support as a Twitchell-Schubert-LowensteinFellow sponsored by the University of Cincinnati.
Selectivity of the Potentiometric Carbon Dioxide Gas-Sensing Electrode M. E. Lopez’ Department of Chemistry, University of Delaware, Newark, Delaware 19716
Experimental and theoretical Investigationsof the potentlometric pCOp electrode have been employed to establish a steady-state model for both organic and Inorganic Interferences at this electrode. It Is shown that electrode response Is governed prlmarliy by the acldlty rather than the volatlilty of the interferents. With the proposed model, quantitative selectivity coefficient values could be calculated In good agreement with experimentally determined values.
The purpose of the present study was to evaluate the usefulness of the response model in predicting the degree of interference observed with the carbon dioxide electrode. The effect of membrane characteristics was also considered. Good quantitative agreement was shown between the theoretically calculated selectivity coefficients and experimentally determined values. Thus, the selectivity of the carbon dioxide electrode was found to depend on the acidity rather than the volatility of the compound. The anomalous response to relatively nonvolatile compounds was interpreted as nonequilibrium behavior.
The selectivity of the carbon dioxide electrode is of particular interest due its widespread use in clinical and industrial analyses. Interference from volatile inorganic and organic acids as well as from nonvolatile compounds has been reported (1-4). The nature of the interference has recently been shown to depend on the characteristics of the gas-permeable membrane (5). The first quantitative evaluation of the potential response of a gas-sensing electrode to interfering compounds was a recent study of volatile amine interference with the ammonia electrode (6). The proposed steady-state response model was based solely on the chemical equilibria in the electrolyte film.
EXPERIMENTAL SECTION Apparatus and Materials. The Orion Model 95-02 carbon dioxide electrodeis the potentiometricgas-sensing electrodewhich was studied. The electrode was assembled with either the Orion carbon dioxide membrane (No. 95-02-04) or the Orion ammonia membrane (No. 95-10-04)with its spacer assembly. The Orion carbon dioxide membrane is polyester mesh supported silicone rubber. The thickness of the silicone rubber (poly(methylvinylsi1oxane))is 0.009 cm and the overall thickness is 0.018 cm. The Orion ammonia membrane is microporous Teflon on a poly(ethy1ene) support. The thickness of the 0.2 wm pore size Teflon is 0.004 cm and the overall thickness is 0.018 cm. Potentiometric and pH measurements were made with a Corning Model 12 research meter and were recorded with a Heath/Zenith Model SR-204 strip-chart recorder. The glass-
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