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Anal. Chem. 1985, 57,2754-2756
Rapid Sub-Picomole Electrochemical Enzyme Immunoassay for Immunoglobulin G W. Uditha de Alwis and George S. Wilson* Department of Chemistry, University of Arizona, Tucson, Arizona 85721
A sandwlch ELISA (enzyme Inked lmmunosorbent assay) that Involves minimum lncubatlon time (0.1 mln) and yet Is sensitive In the femtomole to picomole range has been designed. The Interassay preclslon is *3% and intraassay preclslon Is better than *3%. For the work described a mouse monoclonal antl-bovine Immunoglobulin 0 has been used as a test system. The Instrumentation Involves the use of a flow-through lmmunoreactor coupled to a thln-layer amperometrlc detector. The lmmunosorbent reactor has been shown to be stable up to 3 months of repeated use (at least 500 assays).
The sandwich assay is well-known for its high sensitivity and precision ( 1 , 2 ) compared , to other heterogeneous enzyme immunoassays, but generally requires long incubation periods and a large number of time-consuming and irreproducible manipulations. These assays are customarily carried out in antibody-coated plastic tubes, which show slow protein leakage (3) and irreproducible surface adsorption behavior ( 4 ) . Moreover the nature of the tube makes it almost impossible to totally automate the assay. An alternative to this has been described by Sportsman et al. (5),where a reusable antibody reactor packed with an antibody covalently attached to a solid support is used. The major advantage of this reactor is that it can be used to carry out several hundred assays before losing immunological activity. It is also possible to characterize the antigenlantibody binding surface especially with respect to binding capacity and stability of the antibody so that the optimal conditions for an assay can be readily established. We report here on the determination of mouse anti-bovine IgG which serves as a model system to demonstrate the efficiency of carefully prepared and characterized immunosorbents. EXPERIMENTAL SECTION Mouse anti-bovine IgG and goat anti-mouse IgG were a generous donation from American Qualex International, La Mirada, CA. The monoclonal mouse ascites fluid was further immunoaffinity purified with a Reactigel-6X-bovine IgG column. The goat antibodies were used without further purification. Crystalline bovine IgG (BIgG) (salt free, lyophilized) and glucose oxidase type X were obtained from Sigma Chemical Co, St. Louis MO. Reactigel-6X was obtained from Pierce Chemical Co., Rockford, IL, and was used as recommended by the manufacturer. All other materials were reagent grade chemicals unless specified. Distilled deionized water was used in the preparation of buffers except that the mobile phase was prepared with doubly distilled water filtered through a 0.45-pm filter, and protected from dust. Affinity Purification of Mouse Ascites Fluid. Mouse ascites fluid was filtered through a 0.45-pm filter and was precipitated by addition of a 45% (NH4)2S04solution as described elsewhere (6). After three precipitations the resulting saline solution was applied to a Reactigel BIgG column equilibrated with 0.1 M phosphate buffered saline (PBS) pH 7.4 at a flow rate of 0.5 mL/min. After being washed with the same buffer for 10 min, the bound antibody was eluted with 2 mL of 0.1 M phosphate buffer (PB) pH 2.0. The eluted antibody was collected in vials containing 5 mL of 0.1 M carbonate buffer pH 9.0. The samples
collected were pooled and concentrated in a MicroProdicon (Biomolecular Dynamics, Springfield, OR) negative pressure dialyzer against 0.1 M phosphate buffered saline, pH 7.4. The final concentrations of the antibody was adjusted to 2 mg/mL. The immunogenic activity of the antibody was determined by reapplying 2 mg of the affinity purified anti-bovine IgG to a column of Reactigel-BIgG and washing off any unbound fractions followed by elution of the antibody. The antibody was recovered quantitatively as observed by its absorbance at 280 nm. This observation was considered as an indication that the mouse IgG was close to 100% active. This antibody is of the IgG, subclass. Preparation of the Microreactor. Bovine IgG was immobilized on Reactigel-6X at pH 9.0 for 30 h at 4 "C as recommended by the manufacturer. The coupled yield of the protein was determined by absorbance measurements at 280 nm of the supernatant before and after coupling. An average yield of 4-6 mg/mL settled gel was observed. The gel was washed in the recommended buffers and packed into 0.2 (i.d.1 X 4 cm stainless steel tubing fitted with acid-passivated stainless steel Swagelok HPLC fittings. Preparation of Anti-Mouse IgGGlucose Oxidase Conjugate. The goat anti-mouse IgGglucose oxidase conjugates were prepared with p-benzoquinone (PBQ) as described by Avrameas et al. (7) and subsequently modified by us (8). The final concentration of the conjugate was set at 2 mg/mL in terms of glucose oxidase concentration. Apparatus. The apparatus used for the assay is shown in Figure 1. The two buffers were selected by use of a divertor valve (Autochrome Model SS-20 Autochrom, Milford, MA) and the pump (Beckman-Altex Model 110) was set at 0.5 mL/min. A 30-pL sample of mouse anti-bovine IgG was injected into the flowing stream followed 2 min later by two 75-pL injections of the conjugate spaced 2 min appart. Three 20-wL aliquots of 1% glucose were injected at 3-min intervals. The timing and the injections were controlled by a Waters Associates Model 710B autosampler. By use of a Valcor (Valcor, Springfield, NJ) low dead volume solenoid valve, the flow was diverted through the thin-layer amperometric detector (Bioanalytical Systems, Model BAS LC-4B) at the time glucose injections were being made. An applied potential of 0.8 V is employed so that the hydrogen peroxide produced by the enzyme reaction may be detected. The electrode fouling by the proteins in solution was prevented by covering the working (Pt) electrode with a cellulose acetate membrane (9). The detector output was processed by a Micromeritics Model 740 integrator,peak area being used as the basis of analysis. After each such determination, the antigen-second antibody conjugate was eluted from the column with 0.1 M phosphate buffer pH 2.0. The microreactor was then equlibrated with the assay buffer for 10 min before the next assay was inititated. The sequence of events and their timing are given in Figure 2. Preparation of Standard and Samples. Three types of standards were prepared. In each case the affinity purified antibody (mouse monoclonal IgG) was used. The 2 mg/mL solution was diluted in 0.1 M PBS pH 6.8, normal control serum, unassayed (Ortho Diagnostics, Raritan, NJ) and in 6 g/dL bovine serum albumin in 0.1 M PBS pH 6.8. The unknown concentrations were prepared in the same diluents and were used with matching standards. RESULTS AND DISCUSSION The standards made in 0.1 M PBS DH 6.8 show a linear detection range of over 3 orders of magnitude. The lower detection limit for the assay is in the low femtomole region
0003-2700/85/0357-2754$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
‘-b
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Table I. Assay Results for Bovine IgG
v
-
amt taken,” fmol
buffer
20.00 93.5 185.0 400.0 935.0
21.0 f 0.01 (+5.25%)b 96.5 f 0.01 (+3.2%) 188.0 f 0.01 (+1.62%) 408.0 f 0.02 (+2.0%) 925.0 f 0.05 (-1.17%)
amt recovered, fmol BSA solutionc 20.90 f 0.05 (+4.5%) 96.00 f 0.05 (+2.67%) 187.5 f 0.05 (+1.37%) 404.0 f 0.05 (+1.0%) 928.8 0.1 (-1.0%)
*
a Corresponds to 20-pL sample. *Accuracyerror. BSA concentration, 6 g/dL.
Val”.
Figure 1. Experimental setup for the electrochemical enzyme Immunoassay.
S-Sample
C- Conjugate
G- Glucose
Flgure 4. Timing diagram, from the left injection order depicted by the corresponding letters and the time in minutes in the lower scale.
+
>Anti-Bovine
B o v i i e IgG
>O
(Mouse) igG
A n t i - Nouse(Roat) 196- Glucose Oxidase
Figure 2. Sequence of events for the sandwich assay of bovine IgG. 12.so
10.00
< w
[r
7.90
< Y
5
5.00
CL
2.50
0.00 0. 00
50 00
100 00
FEMTO M O L
I S 0 00
200 00
IGG
Flgure 3. Calibration curve for standards made up in PBS pH 6.8, in the 3-225 fmol/injection region. Correlation coefflcient is 0.976.
from the results shown in Figure 3. The detector response at peak maximum is 10 nA at the lower limit of the assay detection. A SIN ratio of 50 is observed at this level. Thus the immunochemistry and not the mode of detection defines the limit of detection. The correlation coefficient is 0.976 for the results shown. The precision is in the order of *3% and the accuracy is f34%. It was observed that if serial dilutions were employed in the preparation of standards and the glassware was silanized prior to use, then a correlation coefficient of i1-2% could be obtained. At very low concentrations a rapid deterioration of the standards and unknowns was observed. We believe this is due to the adsorption of proteins on glass and their subsequent denaturation.
When the standards were prepared in 6 g/dL bovine serum albumin, which is the approximate total protein concentration in serum, there was no appreciable decrease.in the detection limit or change in the correlation coefficient. For a calibration curve generated in the range of 3-205 fmol, a value of 0.9997 was obtained. The precision wm the same as that for an assay where standards were prepared in pure buffer. In the case of control serum, a different situation was encountered. The lower detection limit was about 1 pmol. Below this level significant curvature in the calibration curve is obtained which eventually results in a leveling out of the response. This can be attributed to a competition between two components in the sample for the same site on the immunosorbent. The competing molecule appears to have a €ower affinity for the site than the assay molecule as the line straightens at levels higher than 1pmol. The interfering substance is not involved in the detection itself since all nonreacting materials are swept out of the reactor prior to the electrochemical determination. Therefore the interferent itself is reacting with the antigen weakly or is nonspecifically adsorbing at or near the binding site. The observation that the effect disappears at higher concentrations of the specific protein indicates the former process is most likely causing this phenomenon. Table I shows the determination of parallel unknown samples carried out using this method. The results highlight the higher accuracies for the determinations carried out with standards prepared in buffers and in BSA solutions compared to those prepared in control serum. The cross reaction effects observed in this assay can be easily removed by using reagents screened for cross reactivity or by diluting the serum, both of which are highly viable options. An interesting phenomenon observed in this work was that if a single injection of the conjugate was made (see Figure 4), the correlation coefficients obtained were not as good as when two injections were made, where the total amount of conjugate is the same. This not only made an improvement in the correlation coefficient but also extended the linear dynamic range by over an order of magnitude. This is due to increased contact time with the binding surface. We believe that approximately 60-75% of the sites are bound after the first injection. The second injection now increases this figure to 85-95%. It also has to be borne in mind that as the amount of the assay antibody is increased, the mass action effects change. This effect combined with the rate of reaction, which has been shown to be extremely high for antibody-antigen reactions (51, allows the short incubation time, about 10 s in
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Anal. Chem. 1085, 57,2756-2758
this case. At higher concentrations there is barely sufficient conjugate to saturate all the immobilized antigen. With a second injection of the conjugate, the linear dynamic range can be increased by about 40% over a single injection double the size. The two-injection method also appears to decrease nonspecific interactions. An alternative to this would be to decrease the flow rate. However this also increases the probability of nonspecific interactions. Experiments carried out using the same apparatus (8) indicate that the turnover of the enzyme is limited by mass transfer of oxygen to the immobilized conjugate a t higher conjugate loadings. This can be alleviated to a significant extent by the use of oxygenated buffers, which lead accordingly to better detection limits. It should be noted (Figure 4) that three injections of glucose were made to determine the amount of immobilized conjugate. If a calibration curve were prepared by using peak area from the first glucose injection, the correlation coefficient would be 0.900. This peak is approximately the same area as the combined area of peaks obtained for the second and third injections. The correlation coefficient and the accuracy increase when the area of the second or third injection is employed. The higher response for the first injection which is indicative of weakly or nonspecifically bound conjugate which is apparently displaced by the injection of glucose and the passage of hydrogen peroxide through the column. As mentioned before, the reactor regeneration step should be carried out with precise timing. The pH 2.0 buffer causes reversible denaturation of the bound antibody. Renaturation and restoration of the proper protein microenvironment takes time. The time allotted for this process has a strong effect on the precision of the measurement. For example if one waits 10 min, the precision is f2-4%, whereas shortening the time to
5 min would give f5-10% precision. A sandwich assay has been employed to determine 1amol (10) of substance. This involves an incubation time of 3 h. The possibility of improving the sensitivity of our system to this limit is being explored at the present time. This would involve slowing the flow rate, using more highly purified conjugate, increasing the detector sensitivity, and the utilizing oxygen-saturated buffers. ACKNOWLEDGMENT We thank Randy Nielsen for the affinity purified anti-bovine (mouse) IgG and American Qualex International for the generous donation of antibodies. The gift of the Model 740 integrator by Micrometrics is gratefully acknowledged. LITERATURE CITED (1) Magglo, Edward T., Ed. "Enzyme Immunoassay"; CRC Press: Boca Raton, FL, 1980; pp 176-177. (2) Masseyeff, R. Scand. J. Immunol. 1978, 8, (Suppl 7), 83. (3) Chessum, B. S.; Denmark, J. R. Lancet 1978, 8054, 161. (4) Cantarero, L. A.; Butler, J. E.; Osborne, J. W. Anal. Biocbern. 1980, 105, 375-381. (5) Sportsman, J. R.; Liddil, J. D.; Wilson G. S. Anal. Chem. 1983, 55, 771-775. (6) Hurrell, J. G. R., Ed. "Monoclonal Hybridoma Antlbodies, Techniques and Applicatlons"; CRC Press: Boca Raton, FL, 1982; p 51. (7) Ternynck, T.; Avrameas, S. Ann. Immunol. (Park) 1976, 127C, 197-208. ( 8 ) De Alwls, W. U.; Lomen, C. E.; Wilson, G. S., unpublished results, 1985. (9) Slttampalam, G.; Wilson, G. S. Anal. Cbem. 1983, 55, 1608-1610. (10) Imagawa, M.; Yoshltake, S.; Ishikawa, E.; Niltsu, Y.; Urushizakl, I.; Kanazawa, R.; Tachibana, S.; Nakazawa, N.; Ogawa, H. Ciln. Chim. Acta 1982, 121, 277-289.
RECEIVED for review June 3, 1985. Accepted July 15, 1985. We thank the National Science Foundation for financial support.
Barium-Selective Electrodes Based on Neutral Carriers and Their Use in the Titration of Sulfate in Combustion Products M a r k u s W. Laubli, Oliver Dinten, Ern0 Pretsch, a n d Wilhelm Simon* Department of Organic Chemistry, Swiss Federal Institute of Technology (ETH),CH-8092 Zurich, Switzerland F r i t z Vogtle, F r a n k Bongardt, a n d Thomas Kleiner Department of Organic Chemistry a n d Biochemistry, University of Bonn, 5300 Bonn 1, Federal Republic of Germany
Highly ilpophillc, eiectrlcally nbutral Ionophores for Ba2+-selectlve solvent polymeric membrane electrodes have been prepared. Sensors based on such ionophores exhiblt a rejectlon of alkali metal Ions of about 10'. They are suitable for the titration of SO,*- with BaZ+ in nonaqueous systems. More than 100 titratlons may be performed wlth no loss In the electromotive behavior of the electrodes.
Several Ba2+-selectiveelectrodes based on neutral carriers have been described (1-5). They are of considerable interest in the titration of sulfate ions with barium ions (1-6). It has been claimed (3,5) that such sensors have very long lifetimes; however this still remains a limiting factor especially when titrations are performed in nonaqueous solvents. The lifetime of such an electrode depends to a large extent on the lipophilicity of the membrane components (7).In order to achieve
a lifetime of 1year for such sensors to continual use with a flowing aqueous sample, a partition coefficient, K , of the carrier between aqueous phase and membrane phase of about lo5 to lo6 is required (7). The carriers N,N,N',N'-tetraphenyl-3,6,9-trioxaundecanediamide (ETH 231) ( 5 ) and antarox CO-880 (3) used previously have partition coefficients of about 102.6and lo3, respectively. We therefore designed Ba2+-selectiveionophores with considerably higher lipophilicities (8). We now report further representatives of such ionophores, the characteristics of the most attractive sensors based on these ionophores, and their use in the titration of sulfate. EXPERIMENTAL SECTION Reagents. All electrolyte solutions for the potentiometric measurements were prepared with doubly quartz distilled water and chlorides of high purity (pro analysis, E. Merck, Darmstadt, GRF; puriss. p.a., Fluka AG, Buchs, Switzerland). Poly(viny1 chloride), o-nitrophenyl octyl ether, dithiooxamide, L-cystein,
0 1985 American Chemical Society 0003-2700/85/0357-2756$01.50/0
