Anal. Chem. 1998, 70, 260-264
Use of a Quartz Crystal Microbalance To Monitor Immunoliposome-Antigen Interaction Kyusik Yun,† Eiry Kobatake,† Tetsuya Haruyama,† Marja-Leena Laukkanen,‡ Kari Keina 1 nen,‡ and ,† Masuo Aizawa*
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan, and VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
We have used quartz crystal microbalance (QCM)-based real-time biospecific interaction measurement to analyze the binding of immunoliposomes to antigen and examined the use of liposomes as signal-enhancing reagents in competitive QCM immunoassay. For the preparation of immunoliposomes, various amounts of bacterially produced lipid-tagged single-chain antibody against 2-phenyloxazolone were incorporated in phosphatidylcholine liposomes. The immunoliposomes bound specifically to immobilized hapten, and this binding was inhibited by soluble hapten in a concentration-dependent manner. In this competitive assay, antigen could be measured in the concentration range from 10-5 to 10-8 M. Immunoliposomes bearing antibodies on their surface have been used in the targeting vehicles for drug delivery1-3 and gene therapy4,5 but have also found use as signal-amplifying reagents in binding assays,6 especially in fluorescence-based applications.7 Traditionally, immunoliposomes have been prepared by chemically conjugating antibody molecules to lipids, e.g., fatty acids.8-11 These chemical derivations involve covalent coupling of the lipid derivative to appropriately exposed sulfydryl and amino groups in the antibody12 and may lead to heterogeneous product and consequently poor orientation on the surface of the liposome. * Corresponding author. (tel) +81-45-924-5759; (fax) +81-45-924-5779; (e-mail)
[email protected]. † Tokyo Institute of Technology. ‡ VTT Biotechnology and Food Research. (1) Bloemen, P. G.; Henricks, P. A.; Bloois, L.; Tweel, M. C.; Bloem, A. C.; Nijkamp, F. P.; Crommelin, D. J.; Storm, G. FEBS Lett. 1995, 357, 140144. (2) Norley, S. G.; Huang, L.; Rouse, B. T. J. Immunol. 1986, 136, 681-685. (3) Jones, M. N.; Hudson, M. J. Biochim. Biophys. Acta 1993, 1152, 231-242. (4) Wang, C. Y.; Huang, L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7851-7855. (5) Wang, C. Y.; Huang, L. Biochemistry 1989, 28, 9508-9514. (6) Park, J. W.; Hong, K.; Carter, P.; Asgari, H.; Guo, L. Y.; Keller, G. A.; Wirth, C.; Shalaby, R.; Kotts, C.; Wood, W. I. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1327-1331. (7) Suzuki, S.; Watanabe, S.; Uno, S.; Tanaka, M.; Masuko, T.; Hashimoto, Y. Biochim. Biophys. Acta 1994, 1224, 445-453. (8) Nassander, U. K.; Steerenberg, P. A.; Jong, W. H.; Overveld, W. O.; Boekhorst, C. M.; Poels, L. G.; Jap, P. H.; Storm, G. Biochim. Biophys. Acta 1995, 1235, 126-139. (9) Geisert, E. E.; Del Mar, N. A.; Owens, J. L.; Holmberg, E. G. Neurosci. Lett. 1995, 184, 40-43. (10) Zelphati, O.; Wagner, E.; Leserman, L. Antiviral Res. 1994, 25, 13-25. (11) Kung, V.; Redemann, C. Biochim. Biophys. Acta 1986, 862, 435-439.
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Furthermore, subjecting the antibody to the covalent coupling may lead to loss or decrease in the activity of the labeled antibody. Recently, genetic engineering has enabled the creation of antibody fragments that bind the antigen like the intact immunoglobulin molecules but that are smaller and may be engineered in a sitespecific manner for different application. Thus, the biosynthetic machinery for the synthesis of Escherichia coli lipoprotein has been exploited to prepare lipid-tagged antibodies which contain a single glycerolipid moiety at the amino terminus.13 This approach may prove particularly suitable for the preparation of immunoliposomes since potentially deleterious chemical treatment of the antibody is not necessary but the lipid anchor is formed during the biosynthesis.14 Lipid-tagged antibodies prepared in this manner retain their antigen-binding properties and provide liposomes with specific antigen-binding capacity.15-17 The selectivity of immunosensors is based on the specific interaction between antigen and antibody.18 An immunosensor is a device comprising an antigen or antibody species coupled to a signal transducer, which detects the binding of the complementary species. Owing to the extremely high affinity of an antibody to the corresponding antigen, immunoassay techniques in general provide high selectivity. The immunochemical interactions may be directly detected by a change in potential difference, current, resistance, mass, and heat, although such a change is not sensitive enough to accomplish a highly sensitive immunosensor.19 Therefore, a detectable signal is usually generated by the addition of a labeled species. These immunosensors use a separate labeled species that is detected after binding by radioisotopes, fluorophores, luminescent molecules, and enzymes.20 An electrochemical luminescent label has been successfully applied to assemble (12) Martin, F.; Hubbell, W.; Papahadjopoulos, D. Biochemistry 1981, 20, 42294238. (13) Laukkanen, M. L.; Teeri, T. T.; Teina¨nen, K. Protein Eng. 1993, 6, 449454. (14) Keina¨nen, K.; Laukkanen, M. L. FEBS Lett. 1994, 346, 123-126. (15) Laukkanen, M. L.; Alfthan, K.; Keina¨nen, K. Biochemistry 1994, 33, 1166411670. (16) Laukkanen, M.-L.; Orellana, A.; Keina¨nen, K. J. Immunol. Methods 1995, 185, 95-102. (17) Kobatake, E.; Sasakura, H.; Haruyama, T.; Laukkanen, M.-L.; Keina¨nen, K.; Aizawa, M. Anal. Chem. 1997, 69, 1295-1298. (18) Aizawa, M. Handbook of Sensors and Actuators; Elsevier Science: Amsterdam, 1996; Vol. 3 pp 85-97. (19) Aizawa, M. Philos. Trans. R. Soc. London, Ser. B 1987, 316, 121-134. (20) Aizawa, M. Adv. Clin. Chem. 1994, 31 , 247-275. S0003-2700(97)00234-5 CCC: $15.00
© 1998 American Chemical Society Published on Web 01/15/1998
a highly sensitive immunosensor for homogeneous immunoassay.21,22 A quartz crystal microbalance (QCM) is a high-sensitivity sensor to the mass deposited on its surface. Recently, QCM methods have found wide applications in immunoassay.23-25 QCM measures the immunochemical interaction without any chemical labels by monitoring the oscillation frequency change, which ideally is proportional to the analyte concentration.26 The crystal surface is coated with an immunoadsorbent that selectively interacts with the analyte of interest and subsequent binding increases the mass of the coated crystal with a resulting shift of its fundamental frequency of oscillation. This technique has also had much success in the analysis of biotin,27 herbicides in drinking water,28 microbes,29,30 enzymes,31 viruses,32,33 and human cells.34 It is, however, a problem that the QCM immunosensor suffers from low sensitivity since the mass change by antigen binding to the crystal surface is small. To enhance the sensitivity, an immunoliposome has been incorporated into a QCM immunosensing system in this study. We describe the QCM measurement to characterize immunoliposome-hapten interactions using liposomes as signal-enhancing reagents that contain biosynthetically lipid-tagged antibodies on their surface. The immunoliposomes bind specifically to immobilized hapten on the crystal surface, and this binding was inhibited by soluble hapten in a concentration-dependent manner. MATERIALS AND METHODS Materials. Egg yolk phosphatidylcholine and 2-phenyl-4ethoxymethylene-5-oxazolone were purchased from Sigma (St. Louis, MO). n-Octyl β-D-glucopyranoside (OG), 6-aminocaproic acid (CA), and Triton X-100 were from Nacalai tesque (Kyoto, Japan). Bovine serum albumin (BSA) was a product of Nesco Bio (Tokyo, Japan). HiTrap Chelating affinity columns were Pharmacia (Uppsalas, Sweden) products. Preparation of 2-Phenyloxazolone Derivatives. Soluble hapten (6-aminocaproic acid derivative of 2-phenyloxazolone, OxCA) and hapten-BSA conjugate (2-phenyloxazolone-BSA, Ox21BSA) were used as competing ligands. 2-Phenyl-4-ethoxymethylene5-oxazolone is a reactive compound which can be coupled (21) Ikariyama, Y.; Kunoh, H.; Aizawa, M. Biochem. Biophys. Res. Commun. 1985, 128, 987-992. (22) Aizawa, M.; Tanaka, M.; Ikariyama, Y.; Shinohara, H. J. Biolum. Chemilum. 1989, 4, 535-542. (23) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 110, 8623-8628. (24) Grabbe, E.; Buck, R. J. Electroanal. Chem. 1987, 223, 67-78. (25) Thompson, M.; Arthur, C.; Dhaliwal, G. Anal. Chem. 1986, 58, 1206-1209. (26) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. (27) Ma´sson, M.; Yun, K. S.; Haruyama, T.; Kobatake, E.; Aizawa, M. Anal. Chem. 1995, 67, 2212-2215. (28) Guilbault, D.; Hock, B.; Schmid, R. Biosens. Bioelectron. 1992, 7, 411417. (29) Prusak-Sochaczewski, E.; Luong, J. H. Enzyme Microb. Technol. 1990, 12, 173-177. (30) Plomer, M.; Guilbault, G.; Hock, B. Enzyme Microb. Technol. 1992, 14, 230235. (31) Vigmond, S. J.; Iwakura, M.; Mizutani, F.; Katsura, T. Langmuir 1994, 10, 2860-2862. (32) Ko¨nig, B.; Gra¨tzel, M. Anal. Chim. Acta 1995, 309, 19-25. (33) Alberl, F.; Wolf, H.; Ko¨sslinger, C.; Drost, S.; Woias, P.; Koch, S. Sens. Actuators, B 1994, 18, 271-275. (34) Ko¨nig, B.; Gra¨tzel, M. Anal. Chim. Acta 1993, 276, 329-333. (35) Gell, P. G. H.; Harington, C. R.; Rivers, R. P. Br. J. Exp. Pathol. 1946, 29, 267-286. (36) Ma¨kela¨, O.; Kaaritien, M.; Pelkonen, J. L. T.; Karjalainen, K. J. Exp. Med. 1978, 148, 1644-1660.
covalently to proteins with concomitant loss of ethoxymethylene group.35 The Ox21-BSA and Ox-CA were prepared as described earlier.36 Briefly, Ox21-BSA was prepared by stirring 600 mg of 2-phenyl-4-ethoxymethylene-5-oxazolone crystals with 2.5 g of BSA in 50 mL of 0.7 M NaHCO3 for 24 h at 4 °C. After incubation, the mixture was centrifuged for 30 min at 30000g (top of the tube) and then extensively dialyzed against 0.15 M NaCl. It has an optical density maximum at 352 nm, and the molar coefficient of extinction is 32 000. 2-Phenyl-4-ethoxymethylene-5-oxazolone (217 mg, 1 mmol) was dissolved in 6 mL of acetone and 195 mg (1.4 mmol) of 6-aminocaproic acid in 14 mL of 0.24 M NaHCO3. When the two solutions were mixed in a flask immersed in an ice bath, a precipitate was formed. The mixture was stirred for 1 h in an ice bath and then overnight at 20 °C. The precipitate gradually dissolved, indicating formation of phOx-CA. This compound was precipitated by bringing the pH to 3.0, the mixture was filtered, and the precipitate was lyophilized. It has an optical density maximum at 348 nm, and the molar coefficient of extinction is 32 000. Purification of Antibody. Lipid-tagged single-chain antibody against 2-phenyloxazolone containing a C-terminal hexahistidinyl tail for immobilized metal affinity purification (Ox lpp-sc-Fv-H6, where lpp is lipoprotein and scFv is the single-chain Fv antibody) was produced in E. coli HB 101 in shake flask culture and purified as described in ref 15, with the exception that Pharmacia HiTrap Chelation Sepharose columns were used instead of batchwise binding to Ni2+-charged Chelating Sepharose. The protein concentrations of the soluble antibodies were determined by using the measured absorbance value and the calculated molar extinction coefficient at 280 nm. Preparation of Liposomes. Liposomes were prepared by a detergent dialysis method using the cellulose dialysis membrane (cutoff 12-14 kDa) essentially as described previously.17 Pure egg yolk phospholipid (10 mg) was dissolved in 2 mL of 20 mM Hepes buffer (pH 7.4) containing 1% (w/v) OG, with or without purified Ox lpp-scFv-H6. It is the concentration of antibody in the original mixture for detergent dialysis. The detergent was then removed by dialyzing against 20 mM Hepes buffer (pH 7.4) overnight in cellulose dialysis bags with two buffer changes at 4 °C. After detergent removal, the liposomes were collected by ultracentrifugation (150000g, 2 h, 4 °C) and suspended in 1 mL of 20 mM Hepes buffer (pH 7.4). QCM Measurements. The hapten-binding activity of the immunoliposome was analyzed by using QCM (Hokuto Denko, Tokyo, Japan). The crystal is 25.4 mm in diameter and 0.3 mm in thickness. The crystal was coated by gold on both sides (Figure 1A). A large electrode (13.15 mm diameter) is patterned on one side and acts as the ground electrode for the oscillator circuit. A smaller electrode (6.25 mm diameter) is patterned on the opposite side and maintained at a frequency 6 MHz. The cell was set up in a flow system in a water bath at a constant temperature of 25 °C, and connected to a Controller HQ-101B (Figure 1B). The cell volume is ∼150 µL. The cell was connected to a 250-µL injection loop and solutions were applied to the flow cell by an intelligent pump 880-PU (Jasco, Tokyo, Japan). The system in liquid phase showed good stability in that the frequency change was 3 Hz even after more than 1 h. Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
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Figure 1. (A) The crystal, fundamental frequency 6 MHz, 25.4 mm in diameter and 0.3 mm in thickness. The crystal was coated by gold on both sides of a large electrode (13.15 mm diameter) and a smaller electrode (6.25 mm diameter). (B) The QCM flow system. The cell was set up in a flow system in a water bath at a constant temperature of 25 °C and connected to a Controller HQ-101B. The cell volume is ∼150 mL. The cell was connected to a 250-µL injection loop, and solutions were applied to the flow cell by a pump.
The QCM measurement for the determination of analytes was performed as follows. The Ox21-BSA was immobilized on the gold surface of a crystal overnight at 4 °C using flat Petri dishes. Ox21-BSA was spontaneously adsorbed overnight onto the evaporated gold surface of the quartz crystal. It was set in the QCM cell; nonspecific binding was blocked by injection of 1% BSA in phosphate-buffered saline (PBS) (pH 7.4). PBS was pumped into the cell at a 1.2 mL/min flow rate. The immunoliposome was incubated with various concentrations of Ox-CA as a soluble hapten. After incubation for 1 h, the reaction mixture was injected onto a sensing crystal surface. After 2 min pumping buffer, the sample solution was injected during 25 s and the flow was stopped and the frequency change recorded at 30 min. The crystal was regenerated by treating them with Triton X-100 and glycine solution (TX/Gly; 1% Triton X-100, 0.05 M glycine, and 0.15 M NaCl, pH 2.5) for ∼10 min. RESULTS AND DISCUSSION The fusion protein consists of a 20-amino acid signal peptide and N-terminal 9 amino acids of lpp, major lipoprotein of E. coli, variable domains of the immunoglobulin heavy and light domain joined by a linker peptide, and a C-terminal hexahistidinyl tail (Figure 2A). Figure 2B is a schematic outline of the lipid-tagged single-chain antibody (anti-Ox lpp-scFv-H6). Immunoliposomes were bound to the hapten-BSA conjugate immobilized on a crystal surface and were also inhibited by soluble hapten-CA derivatives (Figure 2C). QCM Measurement of Immunoliposome Binding. Figure 3 shows a typical QCM readout of the binding of immunolipo262 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
Figure 2. (A) Schematic drawing of the lipid-tagged antibody. The fusion protein consists of a 20-amino acid signal peptide and N-terminal 9 amino acids of lpp, major lipoprotein of E. coli, variable domains of the immunoglobulin heavy (VH) and light (VL) domain joined by a linker peptide, and a C-terminal hexahistidinyl tail. The resulting lipid-tagged antibody carries a single covalently bound glycerolipid anchor at the N-terminal cysteinyl residue of the polypeptide (signal peptide processed). (B) Schematic outline of the lipidtagged single-chain antibody (anti-Ox lpp-scFv-H6). (C) Schematic representation competing free hapten with Ox21-BSA to single-chain antibody surface-loaded in liposome. Immunoliposomes were bound to the hapten-BSA conjugate immobilized on the crystal surface, and the binding was inhibited by soluble hapten (Ox-CA).
somes to antigen. It shows the binding of Ox lpp-scFv-H6 containing liposomes to Ox21-BSA on a crystal surface coated with gold. After injection of buffer, the stable frequencies (F1) of the crystal, immobilized with Ox21-BSA and BSA, were measured. After the reaction, the crystals were washed by buffer and the stable frequencies (F2) again measured. The frequency change (Fliposome ) F2 - F1) was related to the total amount of immunoliposomes adsorbed onto the crystals. The adsorption of the liposome increases the mass of the crystal surface and decreases proportionally the resonance frequency.37 This resulted in a rapid frequency shift of ∼385 Hz. In this experiment, the crystal used was regenerated by treating them in a TX/Gly solution for ∼10 min by dissolving the liposome, and the frequencies almost returned to their F1 values. By means of such treatments, the crystal could be used repeatedly 10 times without detectable loss of sensitivity. (37) Suleiman, A.; Guilbault, G. J. Am. Chem. Soc. 1991, 201, 58.
Figure 3. Typical readout of the QCM sensors. The binding of immunoliposomes (100 µg/mL antibody in Hepes) to the Ox21-BSA (200 µg/mL in PBS) on the crystal surface was analyzed by QCM measurement. Nonspecific binding was blocked by injection of 1% BSA in PBS. The crystal was regenerated by treating it with TX/Gly. Fliposome ) F2 - F1 (F1/F2, stable state frequency before/after after injection of liposome). The frequency change (Fliposome ) 385 Hz) was related to the total amount of immunoliposomes adsorbed onto the crystals.
Antibody Content of the Immunoliposomes. To measure the response as a function of the antibody content of the immunoliposomes, a binding assay for hapten-conjugated BSA (Ox21-BSA) was performed with liposome preparations containing different amounts of antibody. In this step, identical amounts of Ox21-BSA (200 µg/mL) were incubated overnight on the quartz surface outside of the cell. As Ox21-BSA (200 µg/mL in PBS) was injected onto a new crystal for 30 min, the frequency change was 44.1 Hz. The frequency change was 15 Hz in the blocking of nonspecific binding by injection of BSA in PBS for 30 min. The Ox21-BSA-immobilized crystal was placed in the cell, and the immunoliposomes were injected. The frequency change of each crystal was plotted against the amount of antibody used for preparation of immunoliposome (Figure 4). Liposomes containing no antibody displayed no binding to the Ox21-BSA-coated crystal, whereas incorporation of antibody resulted in QCM frequency changes which increased with the amount of antibody. As a sufficient frequency change could be obtained at 150 µg/mL antibody of reaction, we used that concentration for further experiments. QCM Immunoassay. In order to evaluate how the antigen density on the crystal surface affects the binding of immunoliposome, Ox21-BSA solution was adsorbed in various concentrations on a crystal surface overnight at room temperature, followed by a constant amount of immunoliposomes (150 µg/mL antibody in Hepes) injected into the cell. After washing with PBS to remove nonspecific bound immunoliposomes on the surface, the frequency change of each crystal was recorded and plotted against the concentration of immobilized antigen (Figure 5). The frequency change depends on the immobilized Ox21-BSA concentrations as a sigmoid curve. Saturated binding of immunoliposome was observed over 100 µg/mL of Ox21-BSA. Finally, competitive immunoassay (immobilized Ox21-BSA, free Ox-CA, and lipid tagged single-chain antibody) was performed
Figure 4. Frequency change of each quartz crystal plotted against the amount of antibody used for preparation of immunoliposome. Ox21-BSA (200 µg/mL) was incubated overnight on the quartz surface outside the cell. The Ox21-BSA-immobilized crystal was placed in the cell and blocked nonspecific binding by injection of 1% BSA in PBS for 30 min. The immunoliposomes were prepared with 10 mg of phosphatidylcholine and varying amounts of antibody (average of four measurements).
Figure 5. Binding affinity of immunoliposomes as determined by QCM in the presence of increasing amount of the Ox21-BSA. The Ox21-BSA was adsorbed in various concentrations on a crystal surface overnight at room temperature, following which, immunoliposome (150 µg/mL antibody in Hepes) was injected into the cell. After washing with PBS to remove nonspecific bound immunoliposomes on the surface, the frequency change of each crystal was recorded and plotted against the concentration of immobilized antigen (average of four measurements).
under the optimized conditions as given above. The immunoliposomes (150 µg/mL antibody in Hepes) were mixed with various Ox-CA concentrations in a 1:1 ratio (v/v) and preincubated for 1 h. Then the hapten-bound immunoliposome was reacted with the Ox21-BSA adsorbed on a crystal surface. A standard curve for Ox-CA is shown in Figure 6. The binding of immunoliposomes to the hapten-coated quartz crystal was inhibited specifically by the preincubation with Ox-CA in a concentration-dependent manner. Soluble antigen could be determined in the concentration range from 10-5 to 10-8 M in the optimized assay conditions. This shows that the immunoliposomes were able to recognize and bind Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
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In our previous work,16,17 a competitive immunoassay using the immunoliposomes was reported. In surface plasmon resonance, the sensor chip with a carboxymethylated dextran layer was used to immobilize Ox21-BSA. Ox21-BSA immobilization was carried out with a special Amine Coupling Kit (Pharmacia Biosensor AB). However, Ox21-BSA was spontaneously adsorbed onto the evaporated gold surface of the quartz crystal. QCM measured the liposome-antigen interaction without the use of any chemically treated sensor chips and any chemical labels of to monitor the oscillation frequency change, which was proportional to the analyte concentration.
Figure 6. Competitive immunoassay for Ox-CA. The immunoliposomes (150 µg/mL antibody in Hepes) were preincubated in the presence of varying amounts of Ox-CA as a soluble hapten. The reaction mixture was incubated in Ox21-BSA (200 µg/mL in PBS) immobilized quartz surface, and the binding of immunoliposome was determined by QCM measurement (average of six measurements).
to the appropriate molecular target. Multivalent binding is expected to lead to higher binding avidity. This range is in good agreement with the measured affinity of Ox-lpp scFv for soluble hapten (∼1 µM).38 (38) Takkinen, K.; Laukkanen, M. L.; Sizmann, D.; Alfthan, K.; Immonen, T.; Vanne, L.; Kaartinen, M.; Knowles, J. K. C.; Teeri, T. T. Protein Eng. 1991, 4, 837-841. (39) Kanazawa, K.; Gordon, J. Anal. Chem. 1985, 57, 1770-1771. (40) Lin, Z.; Yip, C. M.; Joseph, I. S. ; Ward, M. D. Anal. Chem. 1993, 65, 15461551. (41) Ko¨sslinger, C.; Uttenthaler, E.; Drost, S.; Alberl, F.; Wolf, H.; Brink, G.; Stanglmaier, A. Sens. Actuators, B 1995, 25, 107-112.
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CONCLUSION In the present paper, we describe the use of immunoliposomes prepared by using genetically engineered lipid-tagged antibodies as signal amplifying reagents in a QCM immunoassay. A liposomal QCM assay offers many advantages: quantitative immunobinding reactions (e.g., clinical chemical analysis) such as real-time monitoring, no need for labeled reagents, high analyte sensitivity and specificity determined primarily by the properties of the antibody-antigen pair, and simplicity of use and cost effectiveness.39-41 ACKNOWLEDGMENT This work was supported in part by the Monbusho International Scientific Research Program (Grant 07044134), funded by the Ministry of Education, Science, Sports, and Culture. M.-L.L. and K.K. are grateful for financial support from Technology Development Center of Finland (TEKES). Received for Review March 4, 1997. Accepted July 30, 1997.X AC970234+ X
Abstract published in Advance ACS Abstracts, December 15, 1997.
