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An immunoassay for chlorinated dioxins in soils
Chemosphere, Vol.19, Nos.l-6, pp 267-270, Printed in Great Britain
1989
0045-6535/89 $3.OO + .00 Pergamon Press plc
AN IMMUNOASSAY FOR CHLORINATED DIOXlNS IN SOILS
Bruce E.Watkins, Larry H. Stanker and Martin Vanderlaan Biomedical Sciences Division, Lawrence Livermore National Laboratory, P.O. Box 5507, Livermore, CA 94550
ABSTRACT An immunoassay for chlorinated dioxins in soils is described. This immunoassay utilizes a competitive ELISA with a dioxin-specific monoclonal antibody and an avidin-biotin amplification system on aqueous solutions obtained after extraction of the matrix and an effective clean-up procedure. It's sensitivity has been shown on oil-soaked, composite California soils spiked with variable amounts of 2,3,7,8-tetrachlorodibenzodioxin. KEYWORDS Dioxin; ELISA; Immunoassay; Soil Analysis; 2,3,7,8-TCDD. INTRODUCTION Effective environmental monitoring of toxic chemicals requires the analysis of a great many samples. The cost and time required to analyze for many chemicals, including chlorinated dioxins and dibenzofurans, by traditional methods often severely limits the scope and thoroughness of a sampling effort. Since most of the samples tested are usually negative, a rapid screening assay is useful to separate the samples which are of no concern from those which require further investigation. Immunoassays are an example of this type of assay (Vanderlaan et aL, 1987). Immunoassays are physical assays which depend on the laws of mass action and should not be confused with biological assays which depend on a biological response. These assays have been widely accepted by clinical laboratories but have yet to be effectively exploited by analytic chemical laboratories. Immunoassays can be rapid, inexpensive, and quantitative with detection limits in the low picogram level. Their potential for parallel processing and automation allows for a high throughput of samples at a modest cost, making possible research and regulatory programs that would otherwise be prohibitively expensive. We have developed a set of monoclonal antibodies which bind with chlorinated dibenzodioxins and dibenzofurans (Stanker et aL, 1987a). Each of these antibodies binds with the various dioxins and furans with unique preferences; and because of these preferences and overall affinity for 2,3,7,8tetrachlorodibenzodioxin (TCDD) one of these antibodies (DD-3) was selected to be used in the immunoassay. Immunoassays for dioxins, the one described here as well as others (Albro et al., 1979; Stanker et aL, 1897b; Vanderlaan et al., 1988), cannot be performed on crude matrices and the dioxins must be extracted and cleaned up to remove interfering substances. We describe here the application of our immunoassay to soils with it's eventual use to aid in the remediation efforts in Missouri.
267
268
METHODS Soil extraction. 10 g of soil were dded by shaking with 10g of sodium sulfate for 1 h. The mixture was shaken with 10 ml of hexane and 5 ml of methanol for 15 min, centrifuged, the hexane layer removed by pipet, and the mixture re-extracted with an additional 10 ml of hexane with 15 min of shaking. We have found that extraction of soils is more efficient if the extraction mixture is vigorously shaken prior to separation of phases (data not shown). This was accomplished by using a commercial paint shaker. Sample clean-uD. The clean-up of soil extracts was as previously described for industrial chemicals (Vanderlaan et al., 1988). Briefly, the combined hexane extracts were evaporated and dissolved in dichloromethane/cyclohexane (1:1) and spiked with Fat Blue B (Hoechst AG). The sample was then applied to a column of activated carbon/glass fibers (1:9), the column was washed with dichloromethane/methanol/toluene (15:4:1) and the dioxins were recovered by reverse elution with toluene. The PCDDs (polychlorinated dibenzodioxins) and PCFDs (polychlorinated dibenzofurans) co-eluted with the colored Fat Blue B. The sample was then applied to a column of fuming sulfuric acid impregnated silica gel/silver nitrate impregnated silica gel, eluted with hexane, and evaporated. ELISA. The residue after the clean-up was then dissolved in assay buffer and directly incorporated into a standard ELISA. The ELISA was performed as previously described (Vanderlaan et al., 1988) except that aliquots of the extract, that were spiked with additional TODD to serve as positive controls, were omitted. Maximum sensitivity was achieved when the amount of coating antigen on the microtiter plate was reduced to 1 ng/well and that the subsequent signal is amplified by use of the ABC kit (Vector Labs, Burlingame, CA). This system utilizes avidin-peroxidase/biotin-antimouse immunoglobulin and results in a highly amplified signal (Hsu et al., 1981). RESULTS Standard curves were developed on extracts of a composite Livermore soil to which an analytic standard of TCDD (Cambridge Isotope Laboratories) was added, The composite soil was a mixture of three different soil types to which had been added 10% w/w spent motor oil to realistically model the composition of the contaminated Missouri soils. The spent motor oil was obtained from a unleaded-fuel burning automobile. The curves shown in Figure 1 represent typical dilution curves generated using analytical standards of TCDD diluted directly into assay buffer and curves when the TCDD was spiked into 1.0 and 0,1 g equivalents of composite soil extract after purification of the extract. The effectiveness of the clean-up procedure is evident from the coincidence of these standard curves.
1,0 0.8 f
0.6 !50% Inhibition f o ,0 .C
=
0.4 I 0.2
0.0
.01
•
.1
1
TCDDstandard(ng perwell)
I mmmmm
10
Fig. 1. Three standard curves of ELISA analysis of TODD in assay buffer ([]), 0.1(•) and 1.0 g ( . ) equivalents of soil per well.
269
The factor which seemed to limit the sensitivity of the previous assay was the amount of proteinhapten conjugate that could be detected by an antibody/enzyme conjugated second antibody detection scheme (Vanderlaan et al., 1988). Considerations of mass action predict that less analyte is required to compete the antibody from the solid phase if there is less hapten attached to the solid phase. In our previous assay the practical limit of detection of the hapten was reached when the amount of protein-hapten conjugate was reduced to 50 ng/well. An ABC reagent kit was evaluated to determine if this avidin-biotin amplification scheme could be applied to this assay. We found that with the ABC system we could reduce the amount of protein-hapten conjugate to 1 ng/well, resulting in a ten fold decrease in the 50% inhibition point of the standard curve from 2.0 to 0.2 ng/well. The immunoassay was next used to evaluate the composite Livermore soil spiked at 0.1, 1.0 and 10 ppb. In this case the TCDD standard was added directly to spent motor oil which was then added to the soil and thoroughly mixed. This spiking method models the manor in which the Missouri soils were contaminated. The soils were extracted and subjected to clean-up described above and the ELISA results are shown in Fig. 2 for analysis of soils at 0.2 equivalents of soil per well. The soil contaminated at 10 ppb should have produced inhibition corresponding to 2.0 ng/well and did register at 1.8 ng/well; the 1 ppb contaminated soil should have registered 0.2 ng/well and did register 0.22 ng/well; and the 0.1 ppb contaminated soil should have registered 0.02 ng/well and did register 0.03 ng/well. 1.0
/e
10 ppb spike < ..J
"' o
0.8 0.6
~.
0.4
¢
0.2
e~
m
0.0
.0
,.1 ppb
jill"
I
I .1 1 TCDD [ng/well]
Fig. 2. Standard curve of ELISA analysis of TCDD in assay buffer and determination of TCDD in spiked soils at 0.2 g equivalents per well. In order to achieve higher sensitivity the soils were also analyzed at 1.0 g equivalent of soil extract per well and the competitive ELISA results are shown in Fig. 3. In this case the 10 ppb contaminated soil was out of the linear region of the ELISA curve and those data are not presented. The 1 ppb contaminated soil should have registered 1 ng/well and did register 2 ng/well; and the 0.1 ppb contaminated should have registered 0.1 ng/well and did register 0.04 ng/well. Neither of the ELISA responses from the 0.1 ppb spiked soils were significantly different from that of uncontaminated soil. DISCUSSION The ability of the clean-up method to remove interfering substances was clearly demonstrated by spiking the samples after extraction and clean-up. This is a distinct improvement over other cleanup methods which were evaluated and failed to remove interfering substances. The assay was also improved in absolute sensitivity by the use of an avidin-biotin signal amplification system. This modified extraction/clean-up procedure is effective in soils and in a variety of other matrices (Vanderlaan et al., 1988) for removing substances which interfere with an immunoassay for chlorinated dioxins. The resulting procedure is an effective screening assay for the presence of subset of the PCDDs and PCDFs, mainly the more toxic tetra- and pentachloro congeners. The amount of TCDD equivalents can be quantitated to +/- 50%, which is typical for immunoassays.
270
1.0 <
0.8
..J W
0.6
_m o
c
0.4 0.2 0.0
.01
.1
1
TCDD [ng/well]
Fig. 3. Standard curve of ELISA analysis of TCDD in assay buffer and determination of TCDD in spiked soils at 1.0 g equivalents per well. This immunoassay is clearly effective in measuring TCDD spikes in one soil type. The utility of this immunoassay can only be evaluated after many incurred samples including a range of soil types have been analyzed. Preliminary data suggest that although the clean-up procedure is effective in removing interfering substances from the soils tested to date, the extraction procedure is quite variable. This variability in extraction efficiently cannot be tolerated in an immunoassay since it is not convenient to add an internal standard for quantitation as is done in analysis by gas chromatography/mass spectrometry. We currently are improving our extraction method.
ACKNOWLEDGEMENTS We would like to thank Ms. Anna Marie D. Adams for her expert assistance in the laboratory. This work was supported by the U.S. Environmental Protection Agency through contract No. DW89931433-01-0 and Hoechst AG and performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48. REFERENCES Albro, P.W., Luster, M.I., Chase, K., Clark, G. and McKinney, J.D. (1979). A radioimmunoassay for chlorinated dibenzo-p-dioxins. ToxicoL AppL PharmacoL 50, 137-146. Hsu, S., Raine, L., and Fanger, H. (1981). Use of Avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. of Histochem. and Cytochem. 29, 577-580. Stanker, L.H., Watkins, B., Rodgers, N., and Vanderlaan, M. (1987a). Monoclonal antibodies to dloxln: Antibody characterization and assay development. J.ToxicoL 45, 229-243. Stanker, L.H., Watkins, B., Vanderlaan, M. (1987b). Development of an immunoassay for chlorinated dloxlns based on a monoclonal antibody and an enzyme Ilnked Immunoscorbent assay (ELISA). Chemosphere 16, 1635-1639. Vanderlaan, M., Watklns, B., and Stanker, L. (1987). Environmental monitoring by immunoassay. Environ. Sci. TechnoL 22, 249-254. Vanderlaan, M., Stanker, L.H., Watkins, B.E., Petrovic, P., and Gorbach, S. (1988). Improvement and application of an immunoassay for screening environmental samples for dioxin contamination. Environmental Toxicology and Chemistry 7, 859-870.
1989
0045-6535/89 $3.OO + .00 Pergamon Press plc
AN IMMUNOASSAY FOR CHLORINATED DIOXlNS IN SOILS
Bruce E.Watkins, Larry H. Stanker and Martin Vanderlaan Biomedical Sciences Division, Lawrence Livermore National Laboratory, P.O. Box 5507, Livermore, CA 94550
ABSTRACT An immunoassay for chlorinated dioxins in soils is described. This immunoassay utilizes a competitive ELISA with a dioxin-specific monoclonal antibody and an avidin-biotin amplification system on aqueous solutions obtained after extraction of the matrix and an effective clean-up procedure. It's sensitivity has been shown on oil-soaked, composite California soils spiked with variable amounts of 2,3,7,8-tetrachlorodibenzodioxin. KEYWORDS Dioxin; ELISA; Immunoassay; Soil Analysis; 2,3,7,8-TCDD. INTRODUCTION Effective environmental monitoring of toxic chemicals requires the analysis of a great many samples. The cost and time required to analyze for many chemicals, including chlorinated dioxins and dibenzofurans, by traditional methods often severely limits the scope and thoroughness of a sampling effort. Since most of the samples tested are usually negative, a rapid screening assay is useful to separate the samples which are of no concern from those which require further investigation. Immunoassays are an example of this type of assay (Vanderlaan et aL, 1987). Immunoassays are physical assays which depend on the laws of mass action and should not be confused with biological assays which depend on a biological response. These assays have been widely accepted by clinical laboratories but have yet to be effectively exploited by analytic chemical laboratories. Immunoassays can be rapid, inexpensive, and quantitative with detection limits in the low picogram level. Their potential for parallel processing and automation allows for a high throughput of samples at a modest cost, making possible research and regulatory programs that would otherwise be prohibitively expensive. We have developed a set of monoclonal antibodies which bind with chlorinated dibenzodioxins and dibenzofurans (Stanker et aL, 1987a). Each of these antibodies binds with the various dioxins and furans with unique preferences; and because of these preferences and overall affinity for 2,3,7,8tetrachlorodibenzodioxin (TCDD) one of these antibodies (DD-3) was selected to be used in the immunoassay. Immunoassays for dioxins, the one described here as well as others (Albro et al., 1979; Stanker et aL, 1897b; Vanderlaan et al., 1988), cannot be performed on crude matrices and the dioxins must be extracted and cleaned up to remove interfering substances. We describe here the application of our immunoassay to soils with it's eventual use to aid in the remediation efforts in Missouri.
267
268
METHODS Soil extraction. 10 g of soil were dded by shaking with 10g of sodium sulfate for 1 h. The mixture was shaken with 10 ml of hexane and 5 ml of methanol for 15 min, centrifuged, the hexane layer removed by pipet, and the mixture re-extracted with an additional 10 ml of hexane with 15 min of shaking. We have found that extraction of soils is more efficient if the extraction mixture is vigorously shaken prior to separation of phases (data not shown). This was accomplished by using a commercial paint shaker. Sample clean-uD. The clean-up of soil extracts was as previously described for industrial chemicals (Vanderlaan et al., 1988). Briefly, the combined hexane extracts were evaporated and dissolved in dichloromethane/cyclohexane (1:1) and spiked with Fat Blue B (Hoechst AG). The sample was then applied to a column of activated carbon/glass fibers (1:9), the column was washed with dichloromethane/methanol/toluene (15:4:1) and the dioxins were recovered by reverse elution with toluene. The PCDDs (polychlorinated dibenzodioxins) and PCFDs (polychlorinated dibenzofurans) co-eluted with the colored Fat Blue B. The sample was then applied to a column of fuming sulfuric acid impregnated silica gel/silver nitrate impregnated silica gel, eluted with hexane, and evaporated. ELISA. The residue after the clean-up was then dissolved in assay buffer and directly incorporated into a standard ELISA. The ELISA was performed as previously described (Vanderlaan et al., 1988) except that aliquots of the extract, that were spiked with additional TODD to serve as positive controls, were omitted. Maximum sensitivity was achieved when the amount of coating antigen on the microtiter plate was reduced to 1 ng/well and that the subsequent signal is amplified by use of the ABC kit (Vector Labs, Burlingame, CA). This system utilizes avidin-peroxidase/biotin-antimouse immunoglobulin and results in a highly amplified signal (Hsu et al., 1981). RESULTS Standard curves were developed on extracts of a composite Livermore soil to which an analytic standard of TCDD (Cambridge Isotope Laboratories) was added, The composite soil was a mixture of three different soil types to which had been added 10% w/w spent motor oil to realistically model the composition of the contaminated Missouri soils. The spent motor oil was obtained from a unleaded-fuel burning automobile. The curves shown in Figure 1 represent typical dilution curves generated using analytical standards of TCDD diluted directly into assay buffer and curves when the TCDD was spiked into 1.0 and 0,1 g equivalents of composite soil extract after purification of the extract. The effectiveness of the clean-up procedure is evident from the coincidence of these standard curves.
1,0 0.8 f
0.6 !50% Inhibition f o ,0 .C
=
0.4 I 0.2
0.0
.01
•
.1
1
TCDDstandard(ng perwell)
I mmmmm
10
Fig. 1. Three standard curves of ELISA analysis of TODD in assay buffer ([]), 0.1(•) and 1.0 g ( . ) equivalents of soil per well.
269
The factor which seemed to limit the sensitivity of the previous assay was the amount of proteinhapten conjugate that could be detected by an antibody/enzyme conjugated second antibody detection scheme (Vanderlaan et al., 1988). Considerations of mass action predict that less analyte is required to compete the antibody from the solid phase if there is less hapten attached to the solid phase. In our previous assay the practical limit of detection of the hapten was reached when the amount of protein-hapten conjugate was reduced to 50 ng/well. An ABC reagent kit was evaluated to determine if this avidin-biotin amplification scheme could be applied to this assay. We found that with the ABC system we could reduce the amount of protein-hapten conjugate to 1 ng/well, resulting in a ten fold decrease in the 50% inhibition point of the standard curve from 2.0 to 0.2 ng/well. The immunoassay was next used to evaluate the composite Livermore soil spiked at 0.1, 1.0 and 10 ppb. In this case the TCDD standard was added directly to spent motor oil which was then added to the soil and thoroughly mixed. This spiking method models the manor in which the Missouri soils were contaminated. The soils were extracted and subjected to clean-up described above and the ELISA results are shown in Fig. 2 for analysis of soils at 0.2 equivalents of soil per well. The soil contaminated at 10 ppb should have produced inhibition corresponding to 2.0 ng/well and did register at 1.8 ng/well; the 1 ppb contaminated soil should have registered 0.2 ng/well and did register 0.22 ng/well; and the 0.1 ppb contaminated soil should have registered 0.02 ng/well and did register 0.03 ng/well. 1.0
/e
10 ppb spike < ..J
"' o
0.8 0.6
~.
0.4
¢
0.2
e~
m
0.0
.0
,.1 ppb
jill"
I
I .1 1 TCDD [ng/well]
Fig. 2. Standard curve of ELISA analysis of TCDD in assay buffer and determination of TCDD in spiked soils at 0.2 g equivalents per well. In order to achieve higher sensitivity the soils were also analyzed at 1.0 g equivalent of soil extract per well and the competitive ELISA results are shown in Fig. 3. In this case the 10 ppb contaminated soil was out of the linear region of the ELISA curve and those data are not presented. The 1 ppb contaminated soil should have registered 1 ng/well and did register 2 ng/well; and the 0.1 ppb contaminated should have registered 0.1 ng/well and did register 0.04 ng/well. Neither of the ELISA responses from the 0.1 ppb spiked soils were significantly different from that of uncontaminated soil. DISCUSSION The ability of the clean-up method to remove interfering substances was clearly demonstrated by spiking the samples after extraction and clean-up. This is a distinct improvement over other cleanup methods which were evaluated and failed to remove interfering substances. The assay was also improved in absolute sensitivity by the use of an avidin-biotin signal amplification system. This modified extraction/clean-up procedure is effective in soils and in a variety of other matrices (Vanderlaan et al., 1988) for removing substances which interfere with an immunoassay for chlorinated dioxins. The resulting procedure is an effective screening assay for the presence of subset of the PCDDs and PCDFs, mainly the more toxic tetra- and pentachloro congeners. The amount of TCDD equivalents can be quantitated to +/- 50%, which is typical for immunoassays.
270
1.0 <
0.8
..J W
0.6
_m o
c
0.4 0.2 0.0
.01
.1
1
TCDD [ng/well]
Fig. 3. Standard curve of ELISA analysis of TCDD in assay buffer and determination of TCDD in spiked soils at 1.0 g equivalents per well. This immunoassay is clearly effective in measuring TCDD spikes in one soil type. The utility of this immunoassay can only be evaluated after many incurred samples including a range of soil types have been analyzed. Preliminary data suggest that although the clean-up procedure is effective in removing interfering substances from the soils tested to date, the extraction procedure is quite variable. This variability in extraction efficiently cannot be tolerated in an immunoassay since it is not convenient to add an internal standard for quantitation as is done in analysis by gas chromatography/mass spectrometry. We currently are improving our extraction method.
ACKNOWLEDGEMENTS We would like to thank Ms. Anna Marie D. Adams for her expert assistance in the laboratory. This work was supported by the U.S. Environmental Protection Agency through contract No. DW89931433-01-0 and Hoechst AG and performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48. REFERENCES Albro, P.W., Luster, M.I., Chase, K., Clark, G. and McKinney, J.D. (1979). A radioimmunoassay for chlorinated dibenzo-p-dioxins. ToxicoL AppL PharmacoL 50, 137-146. Hsu, S., Raine, L., and Fanger, H. (1981). Use of Avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. of Histochem. and Cytochem. 29, 577-580. Stanker, L.H., Watkins, B., Rodgers, N., and Vanderlaan, M. (1987a). Monoclonal antibodies to dloxln: Antibody characterization and assay development. J.ToxicoL 45, 229-243. Stanker, L.H., Watkins, B., Vanderlaan, M. (1987b). Development of an immunoassay for chlorinated dloxlns based on a monoclonal antibody and an enzyme Ilnked Immunoscorbent assay (ELISA). Chemosphere 16, 1635-1639. Vanderlaan, M., Watklns, B., and Stanker, L. (1987). Environmental monitoring by immunoassay. Environ. Sci. TechnoL 22, 249-254. Vanderlaan, M., Stanker, L.H., Watkins, B.E., Petrovic, P., and Gorbach, S. (1988). Improvement and application of an immunoassay for screening environmental samples for dioxin contamination. Environmental Toxicology and Chemistry 7, 859-870.