Environ. Sci. Technol. 2000, 34, 1603-1608
Development of Robust, Environmental, Immunoassay Formats for the Quantification of Pesticides in Soil GILLIAN STRACHAN,† JULIE A. WHYTE,† PETER M. MOLLOY,† GRAEME I. PATON,‡ AND A N D R E W J . R P O R T E R * ,† Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, Scotland, U.K., and Department of Plant and Soil Science, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen, AB24 3UU, Scotland, U.K.
The use of antibodies, or more specifically recombinant antibody fragments (scAbs), for the immunodetection of pesticides, could provide a rapid, reliable, and robust method of environmental analysis. The inherent instability of some antibody structures in nonphysiological conditions, however, limits the utility of this approach. We have developed a generic strategy for the stabilization of scAbs in polar and nonpolar solvents, and in a range of other denaturing environments, by the introduction of an interdomain disulfide bond, generating stabilized antibody fragments or stAbs. StAbs specific for the phenoxyacid herbicide mecoprop and the phenylurea herbicide diuron were fully functional when compared to their equivalent unmodified scAbs but significantly more stable in nonphysiological conditions associated with the extraction of organics from environmental samples. Antiatrazine, mecoprop, diuron, and paraquat stAbs were successfully used to accurately identify and quantify pesticides present in solvent and aqueous extracted soil samples. Immunoassay data were validated by HPLC. Recombinant stAbs can be produced readily, cost-effectively, and in quantity from Escherichia coli expression systems and provide robust reagents for immunoassay detection of pollutants present in complex environmental matrices.
Introduction Since the 1940s the need to deter pests and weeds, to achieve greater and greater crop yields, has encouraged the production of several hundred pesticides of diverse chemical nature. Herbicide classes include the triazines, phenoxyacids, and phenylureas. However, by the late 1970s the public was becoming concerned about the leaching of pesticide residues and metabolites into surface groundwater, contaminating drinking water supplies. A concern that is still present today (1). * Corresponding author phone: + 44 (0) 1224 273170; fax: +44 (0) 1224 273144; e-mail:
[email protected]. † Department of Molecular and Cell Biology, University of Aberdeen. ‡ Department of Plant and Soil Science, University of Aberdeen. 10.1021/es991053n CCC: $19.00 Published on Web 03/17/2000
2000 American Chemical Society
Many methods exist for quantifying pesticides present in water and soil samples. Most involve chromatographic analytical procedures (gas or liquid) utilizing a range of detectors. These methods are time-consuming and require highly trained operators (2, 3). In many instances, the use of antibodies for immunodetection of pesticides offers a superior alternative (4). Immunoassays have been used for the last 30 years in medical diagnostics and have more recently been applied to the detection of pesticides in environmental matrices (5-7). Monoclonal or polyclonal antibodies have also been used for analysis of pesticides in complex matrices such as soil and food (8-10). The development of recombinant antibodies consisting of just the antigen binding domain (Fab) or the antibody variable domains (Fv) of whole antibodies is slowly replacing the traditional use of monoclonal antibodies (for reviews see refs 11 and 12). These antibody fragments can be generated much faster than whole antibodies via traditional hybridoma technology and can be produced in larger quantities (12). A number of functional antibody fragments specific for herbicide targets have now been expressed and characterized, and the quantification of trace amounts of pollutants from aqueous sample extracts demonstrated (13-15). In traditional environmental assay formats, these antibody fragments show significantly improved sensitivity over the monoclonal antibody from which they were derived (15, 16). To date, antibody fragments have had limited application to the analysis of pesticides present in soil, unless aqueous extraction procedures were first used (11). Many standard soil extraction methods require incubation with methanol or other solvents (17). Antibody fragments remain as stable as monoclonal antibodies in methanol concentrations below 10% but denature readily above this concentration (18-20). Single chain antibody fragments (scAbs) have been made more stable in polar and nonpolar solvents, and in a range of additional denaturing environments, by the introduction of an interdomain disulfide bond, generating stabilized antibody fragments or stAbs (20). In this study we have demonstrated that the addition of a disulfide bond may provide a generic approach to the stabilization of antibody fragments specific for other pesticide classes including the phenoxyacids and phenylureas. In addition, stAbs selective for atrazine, mecoprop, diuron, and paraquat have been used to accurately quantify pesticide levels present in solvent and aqueous extracted soil samples.
Experimental Section Plasmids and Bacterial Strains. Antibody fragment expression was achieved using the dicistronic, expression vector pIMS147 which is modified from pHELP1 (21), with expression of an antibody fragment cassette and skp gene under the control of the tac promoter. The antibody fragment contains a 14 amino acid linker, a human CK domain fused to the 3′ end of the variable domains and an affinity tag of six histidine residues inserted 3′ of the CK domain. Because the expressed protein includes the human CK domain the antibody fragment is referred to as a scAb to distinguish it from a scFv fragment (22). Coexpression of SKP protein has been shown to improve functional yields by reducing the toxicity of antibody fragment expression (15, 21). All vectors were transformed and expressed in Escherichia coli strain XL-1 Blue (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1, lac[F? proAB, lacIqZA¨ M15, Tn10 (tetr)]). Construction of Disulfide Stabilized Fragments. ScAbs specific for paraquat, mecoprop, and diuron (13, 15, 23) were VOL. 34, NO. 8, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic representation of the (a) scAb, (b) SS1 stAb, and (c) SS2 stAb fragments. VH and VL are the antibody fragment variable heavy and light domains, respectively. The dashed line (- - - -) connecting the VH and VL represents a 14 amino acid flexible linker peptide (27). CK is the constant domain of a human light chain (27), and 6-His is a histidine tail (22) which together allow quantification and purification of the antibody fragments, respectively. Amino acid residues (b) substituted with cysteine (Kabat numbering, 25). used as the starting material for the generation of stAbs. Details of the method of stabilization has been previously described (20) for an antibody fragment against the pesticide atrazine. An interdomain disulfide bond was encouraged to form between the variable heavy (VH) and light (VL) domains in each antipesticide scAb by mutating a single amino acid in the VH and VL to a cysteine residue using a QuikChange Site-Directed Mutagenesis protocol (Stratagene) and two synthetic complementary DNA primers. The introduction of cysteines at positions H44, L100 created pIMS147-SS1-His (expressing SS1 stAb) and at H105, L43 created pIMS147SS2-His (expressing SS2 stAb) (Figure 1). The residues chosen are conserved in most antibodies and predicted to allow stabilization without affecting antigen binding (24). Residue nomenclature is based on Kabat numbering (25). Growth and Expression of Bacterial scAb. Using a method based on published protocols (26) single E. coli XL-1 Blue colonies each containing different pIMS147 versions were grown overnight in 5 mL of LB containing 1% (w/v) glucose, 50 µg/mL of ampicillin (amp), and 12.5 µg/mL of tetracycline (tet) at 37 °C. This culture was used to inoculate 50 mL of Terrific Broth (TB), containing 1% glucose, 50 µg/mL of amp, and 12.5 µg/mL of tet, in 250 mL baffled flasks, and the culture was grown for 16 h at 37 °C and 250 rpm. These cultures were pelleted at 4000 rpm and 4 °C for 20 min and then resuspended in 50 mL of fresh TB (containing additions as above) and grown overnight at 25 °C and 250 rpm. At an OD650 nm of approximately 15 the cells were pelleted as before and resuspended in fresh TB containing 50 µg/mL amp and allowed to recover for 1 h at 25 °C before induction of antibody expression with 1 mM final concentration isopropylthio-βD-galactosidase (IPTG), for 4 h. Cells were once again pelleted, and the pellet was resuspended in 1/10 volume of 200 mM Tris-HCl , 20% Sucrose pH 7.5, 1 mM EDTA, 500 µg/mL of lysozyme and incubated with gentle shaking on ice for 15 min. An equal volume of ice-cold 5 mM MgSO4 was added, and the incubation continued for 15 min. The suspension was pelleted at 13 000 rpm and 4 °C for 20 min, and the 1604
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supernatant containing the periplasmic fraction was harvested, ready for purification. Antibody Fragments Purification and Quantification. All antipesticide scAbs and stAbs were purified via the 6-His tail using Ni2+ charged immobilized metal affinity chromatography (IMAC) Fast Flow Sepharose resin (22). The presence of scAb was detected by capture ELISA of the human CK domain (27). The scAb concentration was calculated from a standard curve plotted using human IgG at known concentrations and corrected for differences in molecular weights between IgG (150 kDa) and scAb (40 kDa). Indirect Competition ELISA. Using a method based on published protocols (16) 96-well, flat bottom Immunolon 4 ELISA microtiter plates (Dynatech Laboratories Ltd) were coated with 100 µL/well of either diuron-bovine serum albumin (BSA), mecoprop-keyhole limpet haemocyanin (KLH), or paraquat-BSA conjugate and diluted in phosphate buffered saline (PBS) to give approximately 2 ng of conjugate per well. The plates were incubated at 37 °C for 1 h and then washed with PBS, 0.5% Tween 20. The plates were blocked for 1 h at 37 °C with 200 µL per well of 2% BSA in PBS and then washed as above. A decreasing range of free diuron, mecoprop, or paraquat concentrations (100 µL) were premixed with 100 µL of purified stAb or scAb (all diluted in PBS) and incubated for 1 h at 4 °C before addition to ELISA plate. The plates were incubated for 1 h at 4 °C and then washed as above before the addition of 100 µL per well of 1:1000 dilution of antihuman free and bound peroxidase conjugate (Sigma A-3682) and incubation at room temperature for 1 h. The plates were then washed and developed using tetramethylbenzidine dihydrochloride (TMB) tablets (Sigma T-3405), according to manufacturers instructions. Absorbance was read at 450 nm (16). Analysis of Stability of Antibody Fragments. Stability analysis was conducted using an ELISA format (20). A 96well, immulon 4 microtiter plate was coated either with diuron-BSA, mecoprop-KLH, or paraquat-BSA conjugate as above. Samples of each of the scAbs and stAbs were prepared at final concentrations of 10 nM, and 100 µL of each was incubated with 100 µL of the test substance. Samples tested in urea (0.2-5 M), methanol (2.5-50% v/v), DMSO (2.550% v/v), or Brij 35 (0.1-5 g/L) final concentration were preincubated for 3 h at 4 °C, and thermal stability samples were preincubated for 3 h at the desired temperature (4-70 °C) before addition to the ELISA plate. The samples in the Pronase (0.01-0.25 g/L) stability assay (resistance to protease attack) were not preincubated before application to the plate. Negative controls were also included to ensure that the conjugate was not degraded during the test. All assays were carried out in triplicate and repeated at least once. Addition of Pesticides to Soil. Field moist soil from Insch, Scotland, U.K. (Dystrochrept sandy loam, 4% organic matter) was collected and passed through a 4 mm sieve, and visible plant material was removed. Field moisture content was determined. Two and a half milliliters of each pesticide, atrazine (30 mg/L), diuron (40 mg/L), mecoprop (500 mg/L), and paraquat (500 mg/L) were mixed, pipetted, and then stirred into 125 g ((0.02 g) of wet weight soil and allowed to equilibrate for 24 h before being aliquoted into five replicates of 25 g samples. These were then left in darkened conditions at 15 °C for 1 week before the pesticides were extracted. Soil water content was maintained gravimetrically at 80% throughout the course of the experiment. Aqueous and Solvent Extraction of Pesticides from Soil. To 0.63 g ((0.01) of soil was added 0.5 mL of ddH2O, and the sample was vortexed for 30 s. The soil samples were agitated overnight in an over and under turntable prior to centrifugation (13 000 rpm) for 5 min to remove the aqueous pesticide solution for analysis.
Methanol (Rathburn HPLC Grade) (20 mL) was added to 10 g ((0.02 g) of soil and sonicated for 5 min. The samples were then rotated and centrifuged as above. This method produced results that correlated closely with methanolSoxhlet extraction for the target pesticides (data not shown). This technique was selected because it is quicker, more automated, and enables a faster throughput of material complementing the benefits of the immunoassay. The aqueous and solvent extracted samples were analyzed for the presence of pesticide using stAb competition ELISA and HPLC. HPLC Analysis of Pesticides. The method used was comparable to U.S. EPA standard techniques (28). HPLC was carried out on a Thermoseparation Spectra Series automated system with a PV5 solvent delivery system. The column consisting of a C18 reverse phase (5 µm, 250 mm × 4.6 mm) Econosphere (Alltech) was maintained at 25 °C. The samples were eluted with 60:40 acetonitrile:water (both Rathburn) and detection was at 210 nm.
Results Expression Levels and Function Analysis of Recombinant scAbs and stAbs. The scAb and both stAbs (SS1 and SS2) (Figure 1), recognizing each of the different pesticides, were expressed, purified via their 6-His tail and expression levels determined by CK ELISA. The addition of two cysteine residues into the variable domains of the antidiuron and antimecoprop stAbs, encouraging disulfide bond formation, did not effect recombinant expression yields measured at 1.5-2 mg/L culture volume. In contrast, the antiparaquat stAb expression appeared more toxic to bacteria yielding only 400 µg/L, while scAb levels were again 1.6 mg/L. The functionality of each scAb and stAb for pesticide conjugate was determined using binding ELISA and surface plasmon resonance analysis (BIAcore 2000). BIAcore analysis confirmed that each stAb fragment was able to bind immobilized conjugate with the same specificity and affinity as the native scAb fragments (results not shown). The specificity of the scAb and the antipesticide stAb fragments for free pesticide was studied further using an indirect competition ELISA. For simplicity, the data presented in each case (Figure 2) compare the native scAb and the “best” performing stAb orientation. A free diuron concentration of 170 and 185 nM reduced the antidiuron scAb and SS2 stAb ELISA signal by 50%, while 40 and 38 nM free mecoprop was required to reduce the antimecoprop scAb and SS2 stAb ELISA signal by 50%, respectively. For the antiparaquat scAb a paraquat concentration of 16 nM reduced the ELISA signal by 50%, while 15 nM free paraquat was required to reduce the signal SS2 by the same amount. Clearly, the introduction of the disulfide bond has had no effect on the ability of the stAbs to bind free pesticide. The SS2 orientation appears better than SS1; however, this improvement over the SS1 orientation was relatively small (results not presented). Stability in Organic Solvents. In general, the improvement in functionality of the SS2 orientation was also observed when the stability of the recombinant proteins was determined after exposure to polar and nonpolar solvents (Figure 3). The antidiuron scAb lost 50% activity in approximately 26% methanol, while the disulfide linked SS2 stAb retained 100% activity in 10% methanol and 50% of its binding in 50% methanol (Figure 3a). Similarly, the antimecoprop scAb was the least stable of the fragments tested, retaining 50% binding activity in 22% methanol, while the antimecoprop SS2 stAb had almost 100% activity in 20% methanol and retained 50% of its binding in the presence of 40% methanol. In contrast, the antiparaquat scAb and SS2 stAb retained 100% binding in 10% methanol and 50% binding in 33% and 26% methanol, respectively (Figure 3a).
FIGURE 2. Indirect competition ELISA assay. Competition was between free antigen and immobilized conjugate on the plate. Antidiuron scAb (b), antidiuron SS2 stAb (O), antimecoprop scAb (1), antimecoprop SS2 stAb (3), antiparaquat scAb (9), and antiparaquat SS2 stAb (0). The dashed line represents the concentration of free pesticide needed to inhibit the binding of scAb or stAb by 50%. Data are presented as an average of three replicates and is a typical data set from replicated experiments. All values were within 5% of mean depicted value. In the presence of DMSO (dimethyl sulfoxide), the pattern of methanol stability for the diuron and mecoprop fragments was repeated (Figure 3b). IC50 values are presented in Table 1. However, the antiparaquat SS2 stAb and scAb outperformed all other fragment structures retaining 100% activity in concentrations up to 15% DMSO and remained 50% functional in the presence of 48% and 42% DMSO, respectively. Thermostability and Stability in Detergents, Proteases, and Denaturants. Data are presented as IC50 values for the most stable fragment orientation (Table 1) following its exposure to a series of different nonphysiological environments. Also included for comparison (Table 1) are data from a similarly stabilized antiatrazine antibody fragment (20). Antidiuron and mecoprop stAbs consistently outperformed their equivalent scAb in all conditions tested, except the presence of detergents where activity was the same. This pattern of results was not observed for the antiparaquat fragments. In this case, the scAb and stAb outperformed all other fragment structures tested including the antiatrazine stAb (20). However, the antiparaquat scAb appeared slightly more stable than the equivalent stAb. Quantification of Pesticides from Soil using stAb and scAb Fragments. To demonstrate the potential of stAbs as “tailor-made” immunoassay reagents for the quantification of pesticide extracted from complex environmental matrices, three stAbs specific for atrazine, mecoprop, and diuron and a scAb fragment specific for paraquat were used to identify and quantify the presence of pesticide in soil water and methanol extracts (Table 2). In general, percentage recovery VOL. 34, NO. 8, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Temperature and Concentrations of Organic Solvents, Detergents, Urea, and Protease Required To Reduce the Binding of the scAb and stAb Antibody Fragments to Their Pesticide-Conjugate by 50% (IC50) fragment
methanol (%)
DMSO (%)
urea (M)
temp (°C)
BRIJ 35 (g/L)
scAb pSS1 stAb
14 >50
Atrazine 9 0.3 19 3
33 41a
>5 >5
scAb pSS2 stAb
26 50
18 35
scAb pSS2 stAb
22 40
Mecoprop 21 0.4 31 >5
scAb pSS2 stAb
33 26
Paraquat 42 >5 48 >5
Diuron 0.8 2.4
32 37
pronase (g/L) 0.02 0.075
5 5
0.04 >0.25
31 39a
>5 >5
0.02 >0.25
48 46
>5 >5
>0.25 >0.25
a For both the antiatrazine and antimecoprop fragments, the opposite orientation of stabilized antibody showed the greatest thermostability (SS2 and SS1, respectively).
TABLE 2. Immunoassay Quantification of Percent Recovered Pesticide from Soil Samples Extracted with Water or Methanol
pesticide atrazine diuron mecoprop paraquat a
antibody ELISA (% recovered) aqueous methanol 5 ( 0.1 66 ( 2 14 ( 0.5 0.1 ( 0
93 ( 3 84 ( 5 26 ( 1 3(0
HPLC (% recovered) methanol 81( 4 92 ( 6 28 ( 3 NDa
ND, not detected.
from solvent extraction was significantly higher than aqueous extracted values. Where possible, data on efficiency of methanol extraction were validated with HPLC analysis. The levels of paraquat in recovery studies were below the limit of detection for HPLC.
Discussion
FIGURE 3. Stability analysis of antidiuron scAb (b), antidiuron SS2 stAb (O), antimecoprop scAb (1), antimecoprop SS2 stAb (3), antiparaquat scAb (9), and antiparaquat SS2 stAb (0) in a range of (a) methanol and (b) DMSO concentrations. Data are presented as an average of three replicates, and this figure is a typical data set from replicated experiments. All values were within 5% of mean depicted value. ELISA controls were included to confirm no significant effect of solvents on immobilized diuron-BSA, mecopropKLH, or paraquat-BSA conjugates. 1606
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The addition, by site directed mutagenesis, of cysteine residues into the variable domains of recombinant antibody fragments to produce stAbs, may provide a generic approach to the stabilization of less robust antibody structures. Antidiuron and mecoprop antibodies, representative of two distinct classes of herbicide, both showed marked improvements in functionality in a number of different denaturing conditions. However, the stability of antiparaquat antibody fragments was not improved by protein engineering. The unmodified antiparaquat fragment (scAb) demonstrated the highest stability of all antibody structures tested, and where relevant comparisons can be made, significantly higher stability than other published fragments (18, 20, 29). Therefore, the apparent failure of the antiparaquat stAb structures to improve protein stability may not be entirely unexpected. The expression, in E. coli, of different antibody fragments is notoriously unpredictable, with very different functional expression yields often obtained (30). These differences are due to sequence mutations causing variation in the degree of protein misfolding, protein aggregation, and cell toxicity (30, 31). This situation could be further exacerbated in stAbs, by the addition of an extra cysteine pair requiring an additional disulfide bond to be formed (32). The antiparaquat stAbs expressed with the lowest yields. The apparent reduction in stability compared with the antiparaquat scAb may
in part be the result of difficulty in accurately determining functional expression yields by capture ELISA. The capture ELISA method is unable to distinguish between correctly folded and misfolded, nonfunctional protein. All three scAbs were stabilized in two different orientations (SS1 and SS2) by modification of two different pairs of framework residues, conserved in most antibodies and predicted by computer modeling to allow stabilization without affecting antigen binding (24). All six stAbs demonstrated no significant loss of antigen specificity or sensitivity, but the SS2 orientation consistently outperformed SS1 in the majority of nonphysiological conditions tested. This relationship is reversed for the antiatrazine stAb where the SS1 orientation is more stable (20). The exact nature of the stabilizing mechanism is not known; however, the addition of an extra disulfide bond may stabilize in several ways including improved packing at the VH/VL interface (33, 34), maintenance of the hydration shell in nonphysiological conditions (35, 36), and increased rigidity of the protein resulting from a reduction of entropy of the unfolded state (37). StAbs show great potential as reagents for rapid, robust, and reliable quantitation of pesticides present in soil following either solvent or aqueous extraction. In our hands immunoassays were simpler and quicker to perform than HPLC analysis, which gave broad agreement with immunoassay for atrazine, diuron, and mecoprop but lacked the sensitivity to detect paraquat at the levels recovered (17). The lower levels of recovery following aqueous extraction are comparable with recoveries obtained in separate studies (17, 38) and reflect, in part, the differing solubilities of pesticides in water. However paraquat has a permanent cationic charge and although soluble in water, binds tightly to negatively charged components in the soil. In addition, the planarity of the paraquat molecule allows it to fit neatly into clay lattices present in soil. Together, these chemical properties severely limit paraquat extraction using aqueous or organic solvents. Paraquat has been acid extracted from soils and detected by gas chromatography (28), and Van Emon (39) used antibodies to immuno-capture paraquat from glass fiber filters. One future application for recombinant antiparaquat scAbs, which can be produced cheaply and in quantity in E. coli, may be as part of a simple immunoseparation protocol to facilitate the extraction of paraquat from environmental samples. In the work presented here, the increased sensitivity of the antiparaquat immunoassay did allow quantification of paraquat following aqueous extraction and provides an indication of the bioavailable toxicity associated with a sample. Numerous reviews and manuscripts have outlined several theoretical advantages of monoclonal over polyclonal antibodies. Despite these advantages most environmental, antihapten, competition assays, available in both the public and private sector, are based on polyclonal antibodies. There are a number of reasons for this, but in general polyclonal antibodies against small molecules yield immunoassays that are more specific, more sensitive, and more robust than monoclonal based assays. In addition, monoclonals are very expensive to produce and maintain in tissue culture. The technology presented here promises to provide the advantages of monoclonal based assays while over coming many of their disadvantages. Recent developments also include the rapid isolation of monoclonal antibodies from libraries (108) of antibody fragments displayed on the surface of filamentous phage (40). Antibody structures present in these libraries could be modified (41) to contain stabilized forms (stAbs) allowing the direct selection of pesticide-specific binders in the presence of denaturants (eg solvents). Together with the low cost of production of recombinant structures in E. coli, these developments make it possible to employ
antibodies in ways (e.g. online monitoring, in situ remediation) that would be too expensive if one were limited to using monoclonal or polyclonal antibodies produced by traditional methods.
Acknowledgments This work was supported by grants from the U.K. Biotechnology and Biological Sciences Research Council and AstraZeneca. Diuron haptens were kindly provided by Dr. Marvin Goodrow and Prof Bruce Hammock, University of Davis, CA (42), the antidiuron antibody by Prof Alex Karu, University of Berkeley, CA, and the PQXB1/2 antiparaquat antibody (13) and paraquat conjugates from AstraZeneca Pharmaceuticals and AstraZeneca Agrochemicals.
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Received for review September 13, 1999. Revised manuscript received January 13, 2000. Accepted January 18, 2000. ES991053N
