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Anal. Chem. 1987, 59, 2688-2691
in this report (37). Work is in progress to ascertain if a more definitive between the Order Of enantiomers and absolute configurations can be found.
LITERATURE CITED Sims, P.; Grover, P. L. Adv. CancerRes. 1974.2 0 , 165-274. Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-581. Gelboin, H. V. Physiol. Rev. 1980, 6 0 , 1107-1166. Conney, A. H. Cancer Res. 1982, 42, 4875-4917. Yang. S . K.; McCourt, D. W.; Leutz, J. C.; Gelboin, H. V. Science 1977, 196. 1199-1201. Yang, S . K.; Roller, P. P.; Gelboin, H. V. Biochemistry 1977, 16, 3680-3686. Yang. S. K.; Chiu, P.-L. Arch. Biochem. Siophys. 1985, 240, 546-552. Weems, H. B.; Mushtaq, M.; Yang, S. K. Anal. Biochem. 1985, 148, 328-338. Yang, S. K.; Mushtaq, M.; Weems. H. B. Arch. Biochem. Biophys. 1987, 255, 48-63. Mushtaq, M.; Weems, H. B . ; Yang, S . K. Arch. Biochem, Biophys 1988, 246, 478-487. Yang, S. K.; Mushtaq, M.; Weems, H. B.; Miller, D. W.; Fu, P. P. Biochem. J . 1987,245, 191-204. Weems, H. B.; Yang, S. K. Anal. Biochem. 1982. 125, 156-161. Yang, S. K.; Weems, H. 6.;Mushtaq. M.; Fu. P. P. J. Chromatogr. 1984,316, 569-584. Yang, S . K.; Mushtaq, M.; Weems, H. B.; Fu, P. P. J. Liq. Chromatogr. 1988, 9 , 473-492. Yang, S . K.; Mushtaq, M.; Fu, P. P. J. Chromatogr. 1986, 371, 195-209. Weems, H. B.; Fu. P. P.; Yang, S. K. Carcinogenesis, 1986. 7, 1221-1230. Harada, N.; Nakanishi, K. Acc. Chem. Res. 1972.5 , 257-263. Yang, S . K.; Weems. H. B. Anal. Chem. 1984,5 6 , 2658-2662. Wong, L. K.; Kim, W. H.; Witiak. D. T. Anal. Biochem. 1980, 101,
-RA-RR . --.
Mushtaq. M.; Weems. H. B.; Yang. S. K. Biochem. Biophys. Res. Commun. 1984, 125, 539-545. Balani, S.K.; Yeh, H. J. C.; Ryan, D. E.; Thomas, P. E.; Levin, W.; Jerina, D. M. Biochem. Biophys. Res. Commun. 1985, 130, 610-6 . . 16. . ~ Balani. S.K.; van Biaderen. P. J.; Cassidy. E. S.; Boyd, D. R.; Jerina. D. M. J. Org. Chem. 1987,5 2 , 137-144. Yang, S . K.; Fu, P. P. 8/0Chem. J. 1984,223. 775-782. Yang, S . K.; Fu. P. P. Chem.-Biol. Interact. 1984,4 9 , 71-88.
(25) Fu, P. P.; Yang. S. K. Biochem. Biophys. Res. Commun. 1982, 109, 927-934. (26) Kedzierski, B,; Thakker, D, R,; Armstrong, R , N,; Jerina, D, M, Tetrahedron Lett. 1981. -~.2 2 . 405-408. (27) Yang, S.K.; Mushtaq, M.;-Chiul P.-L. I n Po/ycyclic Hydrocarbons and Cancer; Harvey. R. G., Ed.; ACS Symposium Series 283; American Chemical Society: Washlngton, DC, 1985; pp 19-34. (28) Mushtaq. M.; Bao. Z.;Yang, S. K. J. Chromatogr. 1987, 385, 293-298. (29) Armstrong, R. N.; Kedzierski, B.; Levin, W.; Jerina, D. M. J. Biol. Chem. 1981, 256, 4726-4733. (30) Thakker, D. R.; Yagi, H.; Levin. W.; Lu, A. Y. H.; Conney, A. H.; Jerina, D. M. J. Bioi. Chem. 1977,252, 6328-6334. (31) Armstrong, R . N.; Levin, W.; Ryan, D.; Thomas, P. E.; Mah, H. D.; Jerina, D. M. Biochem. Siophys. Res. Commun. 1981, 100, 1077- 1084. (32) Bao, 2.; Yang. S. K. Pharmacologist 1986, 28, 240 (abstract no. 793). (33) Dipple, A.; Moschel, R. C.; Bigger, C. A. H. Chemical Carclnogens, Second Edition, Revisedand Expanded; Searle, C. E., Ed.; ACS Symposium Series 182, American Chemical Society, Washington, DC, 1984; Vol. 2, pp 19-34. (34) Herbstein, F. H.; Schmidt, G. M. J. J . Chem. SOC.1954,3302-3313. (35) Briant, C. E.; Jones, D. W.; Shaw, J. D. J. Mol. Struct. 1985, 130, 167-176. (36) Kashino, S.;Zacharias, D. E.; Prout, C. K.; Carrell, H. L.; Glusker, J. P.; Hecht, S. S. Acta Crystallogr. Sect. C : Cryst. Strucf. Commun. I984 C40, 536-540. (37) Unpublished work. 1987. ~
~~
RECEIVED for review May 18,1987. Accepted August 4,1987. This work was supported by Uniformed Services University of the Health Sciences Protocol R07502 and U. S. Public Health Service Grant CA29133. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, DHEW Publication No. (NIH) 78-23.
Reversibly Immobilized Glucose Oxidase in the Amperometric Flow-Injection Determination of Glucose W. Uditha de Alwis, Brian S. Hill, Bruce I. Meiklejohn, and George S. Wilson* Department of chemistry, University of Arizona, Tucson, Arizona 85721 Glucose oxidase (EC 1.1.3.4) is immobilized in a reactor coupled to a flow-lnjectlon analysls system by using an hmunological reactlon. This reactor can then be used to determlne gh~cosein serum and other samples by electrochemlcal monltorlng of H,02 produced as a result of the reaction of the glucose oxidase catalyzed reactlon of glucose wlth oxygen. The reactor Is packed with a support on whlch an antibody Is immobilzed. An enzyme-labeled antibody conjugate or an Immune complex of antiglucose oxldase-glucose oxidase Is passed over the reactor packing, whlch Immoblilzes the enzyme as a result of the lmmunoioglcal reaction. The reactor can be eluted and reloaded with the conjugate to within f5% of the original actlvlty. The llnear dynamic ranges for the reactors are 1.1 X 10-lo-l.l X IO-' and 1.1 X lO-'-l.l X IO-' mol for the immune complex and conJugate loaded reactors, respectively, for a 20-pL sample. On the basis of these llnear detectlon ranges a triplicate analysis on a 1-FL serum sample can be carrled out. *To whom correspondence should be addressed. Present address: Department of Chemistry, U n i v e r s i t y of Kansas, Lawrence, K S 66045. 0003-2700/87/0359-2688$01.50/0
The use of immobilized enzymes in the determination of substrates is well-documented (1-3). This includes the use of enzyme electrodes ( 4 , 5 )and immobilized enzyme reactors of both open-tube and packed-bed types (6, 7). These latter methods are extremely desirable because they are highly sensitive and low in reagent consumption and the reactors can handle low-volume (microliter) samples. In most reactors where electrochemical sensors are employed, sample pretreatment is necessary to remove proteins, which would otherwise foul the electrodes. This is achieved by the interposition of dialysis membranes or by use of precolumns. The use of dialysis membranes (8)brings about an attenuation of the signal, which can seriously decrease the detection limit, and precolumns (9) can fail due to overloading. In highperformance applications where high specific activity of the immobilized enzyme must be maintained, regeneration or replacement of the reactor may be frequently required. The new reactor may have very different characteristics, hence requiring time-consuming calibrations and equilibration intervals, thus increasing downtime. In addition, separate enzyme reactor columns are required for each assay. In this publication we wish to discuss the indirect immobilization of the enzyme such that regeneration of the system in the case G 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
of a failure requires only simple manipulations. The process takes less than 1 h. The reversible immobilization of enzyme is achieved by utilizing several different reaction sequences involving antibody-antigen reactions. In the first case the enzyme is covalently attached to an antibody and is passed through a packed-bed reactor in which the antigen is covalently immobilized. The enzyme is immobilized due to the antibody-antigen reaction. In the second method a goat anti-mouse Fab' fragment is again immobilized. This support is now used to reversibly immobilize a monoclonal anti-enzyme antibody, which is in turn used to immobilize the enzyme. The advantage of the initial immobilization of the antimouse antibody is that it provides a universal method for the immobilization of any mouse monoclonal antibody. Thus several enzymes could be linked to the surface by using different anti-enzyme antibodies. These methods are examined as a possible approach to the preparation of easily regenerable highly active enzyme reactors by using glucose oxidase as a model system for flow-injection analysis (FIA). EXPERIMENTAL SECTION Human IgG was obtained as "Gamastan" (Cutter Laboratories, Berkeley, CA) at a local drug store and used without further purification. Anti-human IgG (goat) was obtained from Cappel Laboratories, Malvern, PA, and was affinity purified against human IgG immobilized on Reactigel6X. Glucose oxidase type X-S and pepsin (porcine mucosa) 1:60000 was obtained from Sigma Chemical Co., St Louis, MO. Sephadex (3-25 and Sephacryl S-300SF were obtained from Pharmacia Fine Chemicals, Piscataway, NJ. Glycidoxypropyltrimethoxysilanewas obtained from Petrarch Systems, Levittown, PA. Lichrosphere SI-300(10-pm diameter) (E.Merck, Darmstadt, Germany) was obtained from EM Scientific, Gibbstown, NJ. Monoclonal anti-glucose oxidase antibodies (IgGJ were prepared according to established procedures (IO). These antibodies were affinity purified against the glucose oxidase apoenzyme immobilized on Fractogel HW-65F. FMP activated Fractogel HW-65F was a generous donation from American Qualex, La Mirada, CA. All other reagents used were reagent grade unless specified otherwise. All water used was double distilled and the FIA buffers were filtered through 0.45-~m filters and protected from dust. Preparation of Glucose Oxidase-IgG Conjugates. The conjugates were prepared by using p-benzoquinone as described elsewhere ( I I ) . Apparatus. The flow-injection apparatus used in this work has been described elsewhere (12). Preparation of the Goat Anti-Mouse IgG Supports. Goat anti-mouse IgG (50 mg)was diasolved in 0.1 M phosphate-buffered saline (NaC1-PBS) pH 7.4 and dialyzed against 0.1 M acetate buffer pH 4.5 for 24 h at 4 "C. To this 1mg of Pepsin was added and incubated for 16 h at 37 "C. The sample was then redialyzed against 0.1 M NaC1-PBS pH 7.4 for 24 h. The specific F(ab')z fragments were harvested by direct affinity purification against mouse IgG-Sepharose 6MB. The affinity purified F(ab'), fragments were immobilized on FMP activated Fractogel HW-65 (Avidgel) according to the manufacturer's instructions. The gel was washed as suggested by the manufacturer and was then cycled back and forth between 0.1 M NaCl-PBS pH 7.4 and 0.1 M phosphoric acid (elution buffer) for five complete cycles. The gel was then packed into a 0.2- X 4-cm stainless steel reactor fitted with Swagelok l/z-in. stainless steel fittings and connected to the flow system. Preparation of Activated Controlled Pore Glass. CPG (controlled pore glass) SI-300(10 g ) was mixed with 10 mL of freshly prepared chromic acid. The mixture was poured on a medium-porosity glass frit and the chromic acid was removed by using suction filtraction. Hot 1 M HNOB(50 mL) was poured over the beads in 4 aliquots and was followed by 100 mL of distilled water. The wet cake obtained was suspended in 45 mL of distilled water and sonicated for 10 min. The silica suspension was heated to 90 "C in a water bath. The suspension was maintained by overhead stirring. Glycidoxypropyltrimethoxysilane (GOPS) (5 mL) was added to the suspension and the pH was adjusted to 3.0 with 1 M H2S04. The pH was maintained
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at 3.0 with addition of 1 M H2S04for 1 h. The suspension was transferred to a centrifuge tube and centrifuged at 2000g for 5 min. The sample was washed three times in distilled water by adding 50 mL of distilled v ater to the tube and vortexing until the whole sample was resuspended. It was then centrifuged as mentioned abdve. After three washes, the sample was transferred to a large test tube and dried in an oven at 105 OC overnight. The preparation of the aldehydic glycophase was carried out as described by Sportsman et. al (13). The average aldehyde loading was 282 11 pmol/g of dry silica (n = 8). Coupling of Human IgG to the Reactor. The human IgG was dialyzed against 0.1 M carbonate buffer pH 9.5 overnight and the protein concentration was adjusted to stand at 2-5 mg/mL. The solution was added at a 2:l (v/v) ratio to the aldehydic glycophase and the tube was rotated overnight at 4 OC. NaBH, (5 mg) was added over 20 min and the solution was allowed to stand for 1 h at room temperature. The supernatant was filtered out and the CPG was washed with 5 volumes of 0.1 M NaCl-PBS pH 7.4 and was then cycled between the pH 7.4 buffer and pH 2.0buffer five times. The washings were collected and the amount of protein bound was determined by difference. The amount of IgG immobilized is approximately 7-15 mg/g of dry silica. Loading the Enzyme Reactor (Enzyme Conjugate)-Reactor A. The conjugate solution was diluted to obtain a 1mg/mL enzyme concentration (IO). Aliquots of 20 pL of this solution were injected into the reactor. These were followed by three 20-pM injections of 0.1% glucose solution at 3 min intervals. The hydrogen peroxide produced from the enzyme reaction was detected amperometrically. Loading the Enzyme Reactor (Glucose Oxidase-AntiGlucose Oxidase)-Reactor B. The antibody-antigen complex was first formed in solution by adding 2 mg of glucose oxidase to 4 mL of the antibody solution which had a concentration of 0.2 mg/mL. An excess of antigen was used to drive the immunological reaction to completion. A 50-pL sample of this solution was injected into the reactor containing immobilized goat antimouse antibody. This was followed by three injections of 0.1% glucose as in the previous case. Regeneration of the Reactor. The reactor was regenerated by eluting the enzyme from the support by using 0.1 M phosphoric acid pH 2.0. Following this, the reactor was equilibrated with 0.1 M PBS pH 6.8 and reloaded with enzyme. Measurement of Enzyme Activity. Glucose oxidase activity in the soluble conjugates and immunecomplex was measured as recommended by Sigma Chemical Co. (Sigma Technical literature accompaning the material) in IU/mL. The measurements were made in a 1-cm quartz cell placed in a Varian Cary Model 219 instrument fitted with a water-jacketed cell holder. In addition, the same procedure was later carried out in a Roche Diagnostics COBAS BIO centrifugal fast analyzer. For this work the reagent volumes were reduced by a factor of 10. The rest of the protocol was unchanged. All measurements were made at ambient oxygen concentrations.
*
RESULTS AND DISCUSSION Two strategies for the immobilization of the enzyme Glucose Oxidase are shown in Figure 1. Implementation of reactor A requires the preparation of an IgG immunosorbent (Ag) and a conjugate formed by the covalent attachment of anti-IgG (Ab) to the enzyme (E). We have shown previously that several conjugates are produced in the coupling reaction: Ab-E, Ab2-E, and Ab3-E (12). Thus the multiple attachment of the enzyme in the reactor as a result of the antibody-antigen reaction contributes significantly to the immobilization stability. For example human IgG (Ag) in the mobile phase is unable to displace the enzyme from the surface. This suggests that the effective binding constant for the reaction is >lo*. On the other hand, the covalent attachment of the enzyme to the antibody results in a t least 52% decrease in enzyme activity. This measurement was made on the soluble conjugate and is therefore not related to possible immobilization effects. The second immobilization strategy is demonstrated in Figure 1,reactor B. This immunosorbent was prepared with polyclonal goat anti-mouse IgG Fab' fragments. Such an
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 Reactor A
Reactor
B
Immobilized H u m a n IgG
Immobilized A n t i Mouse lgG Fab’ Fragment
Anti-Human IgG Glucose Oxidaso Conjugate
Mouae Anti-Giu. Ox.
- GLU. Ox. Immune Compler
Flgure 1. Immobilization schemes for the enzyme for the antibody conjugate and the immune complex.
immunosorbent can be expected to bind to practically any mouse IgG antibody, thus rendering it a universal immunosorbent when used in conjunction with a monoclonal antibody specific for a particular enzyme. The monoclonal antibody thus becomes a specific “linker arm” between the reaction support material and the enzyme. The reaction of the monoclonal antibody with the enzyme causes less than 2% loss of enzyme activity. Because the monoclonal antibody cannot undergo multivalent interaction with the enzyme, the effective binding constant for the antibody-enzyme reaction is generally correspondingly lower. As will be discussed subsequently, this leads to some enzyme leakage from the reactor. This problem may be corrected by the use of a monoclonal antibody with a higher binding constant. Reactor A showed a decrease of less than 2% activity per day (with daily use) while reactor B showed decreases on the order of 3-5%. Figure 2 shows the loading curve results for successive injections of glucose oxidase. For reactor A, the glucose oxidase is injected in the form of a covalent Ab-E conjugate and in reactor B as an immune complex. The curve for reactor A reaches a well-defined limiting value corresponding to the saturation of binding sites. Subsequent washing of the reactor bed by the flowing buffer solution (0.1 M NaCl-PBS pH 6.8) does not result in significant decrease in signal. This suggests that the initial reaction of the conjugate is strong and very specific. By contrast, the loading of reactor B does not reach a limiting value. If the reactor is washed for 30 min an approximately 20% decrease in signal is observed, after which the signal stabilizes. It appears that the initial reaction is subject to nonspecific reactions, which are not sufficiently stable to keep the enzyme immobilized. I t is possible that the limiting response observed in the Figure 2 loading curve could be due either to a limited number of binding sites on the column or to attainment of a diffusion-controlled limit for enzyme catalysis. To distinguish these two cases, several reactor columns were prepared with exactly the same geometry and the same support material. The only difference between these several reactors is the number of binding sites available. This was established by using previously developed methodology (13). A series of loading curves similar to Figure 2 were obtained, giving limiting responses 2-3 orders of magnitude higher than that shown. This suggests that the limiting response is due to the number of binding sites (i.e. the total possible loading of the enzyme)
Table I. Reproducibility of the Conjugate and Immune Complex Loading peak area in arbitrary units,” Xl06
cycle no.
reactor B
reactor A
1
2.130 2.135 2.129 2.125 2.135 2.132 2.133
2 3 4 5 6
10
f 0.010 f 0.015
6.321 5.902 6.154 6.254 5.985 6.301 5.852
f 0.010 f f f f
0.013
0.012 0.015 0.012
f 0.010 f 0.012 f 0.011 f 0.010 f 0.013 f 0.012 f 0.011
Electrochemical detection of hydrogen peroxide produced from the injection of 1 ng and 1 rg of conjugate and immune complexes, respectively, to reactors A and B followed by three 20-pL injections of 5.5 mM glucose solution. Table 11. Comparison of Data Obtained by the Beckman Astra and Reactor A patient
Beckman Astra’
FIA/reactor A’
1 2 3
67 121 172 283
63.49 f 0.2; 117.1 f 0.25 174.07 f 0.31 285.48 f 0.20
4
Serum glucose mg/dL. Sodium azide ( 2 % J present in sample and assay buffer. Table 111. Recovery of Glucose in Control Serum by Reactor B amount added” 50.0 100.0 150.0 200.0 250.0
amount recovered 50.1 f 0.3 99.8 149.7 201.2 248.3
f 0.2 f 0.2
f 0.3 f 0.2
Samples were prepared by dissolving the appropriate amounts in control serum.
and not to response limited by mass transfer of glucose (or oxygen) to the immobilized enzyme. The data for Tables I-111 were obtained on the rising portion of the loading curve in
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 G L U C O S E O X I D A S E L O A O I N G CURVE 1B.M
.
0.M
1
4. Bo
2.40
GLU.
OX.
7. M
8. @n
U MOLES
Flgure 2. Enzyme loading curves: X, conjugate loading curve; 0,
immune complex loading curve. each case. The loading was tested on the rising part of the curve. This region is more sensitive to small changes in enzyme loading and therefore would be expected to yield degraded precision. However the data given in Table I show the excellent reproducibility of the method. The slope of this region is 3 X 10" area unitslg of conjugate. Therefore it is possible to measure changes in enzyme loading as low as g. Table I shows the results of cycling the reactors by successive loading and elution of enzyme. The precision of subsequent hydrogen peroxide measurements within a given reactor cycle is better than 1%for both the reactors. The precision from cycle to cycle is similarly better than 1% . Calibration curves run on each of the reactors gave dynamic and 1.1 x 10-lo-l.l x linear ranges of 1.1 x 10-9-l.1 x lo4 mol for reactors A and B, respectively. The upper end of the range was defined by the limits of the detector. This problem can be easily corrected either by decreasing the sample size or by sample dilution. The low end of the curve was defined by the detection limit set at a SIN ratio of 10. The limits of detection for the two systems are 50 pmol for reactor A and 12 pmol for reactor B, respectively. The discrepancy between the limit of detection and the lower limit for linear detection range arises as a consequence of the SIN ratio requirements. A transient pressure pulse occurs in the system 1min before each injection. The rest of the base line is perfectly flat. When this transient signal is taken into consideration and the detection limit defined at a S I N of 2, the lower end of the linear range can be extended to the detection limit mentioned above for the two systems. At these concentration levels and a buffer flow rate of 0.5 mL/min the
2691
conversion efficiencies were 33% and 60% for reactors A and B, respectively. the sample throughput was about 20/h. The efficiency can be increased by decreasing the flow rate but this will result in broader peaks and decreased sample throughput. The performance of the two reactors are very similar except for the linear detection range. Both reactors could be loaded, eluted, and reloaded to within f 3 % of the previous activity (Table I). The values obtained for glucose in serum samples show good agreement with those obtained by the Beckman Astra (Table 11). Considering the fact that the method of determination can give rise to somewhat different values for the same sample (Technical literature accompanying SeraChem clinical chemistry control serum (Human) Assayed (product no. 2907-63)),recovery studies were carried out for both reactors with control serum. Both reactors yielded good correlation. Results for reador B are shown in Table 111. The resulte, for both reactors A and B suggest that they can be used for measurements on undiluted serum without displacement of enzyme or interference by serum components.
ACKNOWLEDGMENT We thank American Qualex for the generous donation of materials. Registry No. EC 1.1.3.4, 9001-37-0; D-glucose, 50-99-7; Fractogel HW-65F, 89492-12-6.
LITERATURE CITED (1) Fundamentals of Clinical Chemistry 2nd ed.; Tietz, W. N., Ed.; Saunders, Phiiadelphla, PA, 1976. (2) Bowers, L. D.; Johnson, P. R. Clin. Chem. (Winston-Salem, N.C.) 1981, 27. 1554-1557. (3) Carr, P. W.; Bowers, L. D. Immobliized Enzymes in Analytical and ClinicalChemistry, Fundamentalsand Applications; Wlley, New York, 1980. (4) Notin, M.; Guillien. R.; Nabet, P. Ann. Bioi. Clin. (Paris) 1972, 30, 193-196. (5) Guilbault, G. G.; Lubrano, G. J. Anal. Chim. Acta 1972, 6 0 , 254-256. (6) Garber, C. C.; Feldbruegge,D.; Miller, R. C.; Carey, R. N. Ciin. Chem. (Winston-Salem, N.C.) 1978, 24, 1186-1190. (7) Dahodwala. S. K.; Weibel, M. K.; Humphrey, A. E. Biotechnol.Bioeng. 1978, 78, 1679-1649. (8) Yao, T.; Sato, M.; Kobayashi, Y.; Tamotsu, W. Anal. Chim. Acta 1984, 765, 291-296. (9) Leon, L. P.; Chu, D. K.; Snyder, L. R.; Horvath, C. Clin. Chem. (Winston-Salem, N.C.) 1980. 26, 123-129. (10) MonoclonalHybridoma AntiTechniques and Applications; Hurreil, J. G. R., Ed.; CRC Press: Boca Raton, FL, 1982. (11) de Aiwis, Uditha; Wilson, G. S., unpublished results, University of Arizona, 1985. (12) de Aiwis, Udltha; Wilson, 0. S. Anal. Chem. 1985, 57, 2754-2758. (13) Sportsman, J. R.; Wilson G. S. Anal. Chem. 1980, 52, 2013-2018.
RECEIVED for review January 27,1987. Accepted July 14,1987. We thank the National Institutes of Health (Grant DK 30718) for financial support.
