Chapter 6
Development of Immunoassays for Detection of Chemical Warfare Agents
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David E. Lenz, Alan A. Brimfield, and Lara A. Cook Biochemical Pharmacology Branch, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5425
With the advent of enzyme linked immunoabsorbent assays (ELISA) and monoclonal antibodies, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman when used in a competitive inhibition enzyme immunoassay (CIEIA) were capable of detecting soman in buffer solutions at a level of 10- M (~180 ng/mL). These antibodies were found to be highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies extended the level of sensitivity to ~80 ng/mL These antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. These reagents offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances. More recent efforts have focussed on developing antibodies specific for sulfur mustard a highly reactive vesicating agent 6
The standard methods of analysis for detection of organophosphorous chemical warfare agents either require time-consuming isolation and cleanup procedures and expensive analytical equipment such as gas chromatography (GC) or gas chromatography-mass spectrometry (GC/MS), or they rely on rather nonspecific color reactions that result from changes in the activity of the enzyme acetylcholinesterase. The former procedures are quantitative but slow and expensive; while the latter rapid approach is rapid, qualitative or semi-quantitative but can give ambiguous results. What would be most desirable is a method that could detect a specific chemical warfare agent in a rapid, quantitative manner. In addition, the results should be subject to minimal interference from hydrolysis products or structurally related compounds. Such a method would have value not only for detecting exposure to a toxic material, but it could also be used to diagnose the type of toxicant so appropriate medical treatment could be administered.
This chapter is not subject to U.S. copyright. Published 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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Antibodies as Analytical Reagents
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One approach that provides many of the features of such an analytical method is based on the use of antibodies specific for the analyte of interest. This has shown considerable applicability in the analysis of insecticides in environmental samples (76) and also has application in clinical and forensic settings. For example, Hunter and Lenz reported that paraoxon, the active metabolite of parathion, could be detected at a level of 1 nM in biological fluids (3). Antibodies as Reagents for Detecting Chemical Warfare Agents. More recent efforts have expanded this approach and applied it to developing antibodies against various chemical warfare agents. Initially, polyclonal and subsequently monoclonal antibodies were produced against the highly toxic organophosphorus chemical warfare agent soman (7-9). These proved to be useful in the establishment of several immunoassays capable of quantitatively determining the amount of soman present in aqueous solution (7) as well as mammalian serum and milk (70, 77). In all cases a competitive immunoassay was the method of choice for quantification. More recently antibodies specific for a variety of chemical warfare agents to include polyclonal (72) and monoclonal (73) antibodies against VX, polyclonal antibodies against sarin (14-16) and polyclonal (17) and monoclonal antibodies (18) against sulfur mustard and sulfur mustard DNA adducts (19,20) have been developed. Considerations for Development of an Immunoassay An immunoassay, to be most effective, should be designed to detect the analyte of interest (the hapten) and, ideally, nothing else. While this may seem obvious, it must be remembered that other analytical approaches such as GC, HPLC or GC/MS often detect a host of species from which the analyte of interest must then be uniquely identified. As with most analytical techniques, it is useful to estimate the expected concentration of the analyte in the milieu chosen for analysis. In the case of immunoassays, if reasonable estimates can be made regarding the concentration and binding constant characteristics of the antibody being developed, then theoretical calculations can be carried out to determine if an assay will have the necessary sensitivity to detect the analyte of interest. Immunogen Development. Since most small molecules (2 p M . In both cases, the level of sensitivity was in excess of that needed to identify the presence of sulfur mustard at concentrations below those that are estimated to cause a vesicant injury on human skin. Immunization of mice with N7-HETE-GMP coupled to keyhole limpet hemocyanin resulted in several hybridomas that produced monoclonal antibodies that recognized the N7-HETE-Gua. Using these antibodies in a CIEIA gave sensitivity comparable with that using the rabbit serum (20). Summary Current efforts have demonstrated that monoclonal and polyclonal antibodies that are specific for three of the chemical warfare organophosphorous nerve agents, soman, sarin and V X can be developed. A l l of these antibodies have been investigated to a sufficient degree to determine if they could be used in an immunoassay to detect the presence of soman, sarin or V X respectively in an aqueous sample. In the case of soman and sarin, the detection limits were in the micromolar range which, while fairly sensitive, would only be able to detect about twice the estimated concentration that would be expected following exposure to an LD50 dose of soman in humans and just equal to the estimated L D dose of sarin in humans (Table II). The antibodies for V X have not yet been utilized in an immunoassay, but to be useful they should also be capable of detecting V X in the sub micromolar range. Based on the reported binding of V X in the n M range (75), these antibodies should be capable of being developed into an assay with sufficient sensitivity to detect V X at concentrations less than those expected after exposure to a toxic dose. In most of the reports to date, the total assay time was two hours or less with an incubation time of the antibody with the nerve agent of 10 to 30 minutes. The anti-soman, anti-sarin and anti-VX antibodies all had the required specificity and exhibited a little or no cross reaction with either the metabolites or hydrolysis products of the respective nerve agents. 5 0
In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
6. LENZ ET AL.
Immunoassays for Detection of Chemical Warfare Agents
Table III. IC50 Values for Inhibitors of Sulfur Mustard antibodies Inhibitor
Structure
G . Sulfur Mustard (HD)
ci
- CH - CH - S - CH - CH -
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2
2
2-Chloroethyl Methyl Sulfide (CEMS)
2
CI - C H - C H - S - C H 2
2
a
4
1.00 X 10" M
5
7.85 X 10r M
5
4
3.92 X 10* M
3
O II 3
7.65 X 10* M
HO - C - C H - (CH ) - a 2
O II HO - C - ^
K . 4-(2-Chloroethyl) Benzoic Acid (4-CBA)
2-2 Thiodiethanol (TDG)
M . 2-Hydroxy ethyl Disulfide (HED)
2
CI - CHj - C H - S - C H
8-Chloro-Caprylic J . Acid (8CCA)
L.
2
2
H . 2-Chloroethyl Ethyl Sulfide (CEES) I.
IC50
^
2
6
- CH - C H - CI 2
2
HO - C H - C H - S - C H - C H - OH 2
2
H M
n U
"
r c t l
u
* "
2
2
CH - S - S - CH - CH -OH 2
2
2
3
4.57 X 10" M
2
> 1.00 X 10" M
2
> 1.00 X 10 M
In the case of antibodies developed direcdy against sulfur mustard, neither the polyclonal nor the monoclonal antibodies (17,18) have the specificity needed for a useful immunoassay (Table III). The antibodies against the N7-HETE-Gua developed by Benschop and co-workers (19-20) do have very high level of sensitivity for the N7-guanine adduct that occurs following the reaction of sulfur mustard with D N A . In this case the authors indicate that they are in the process of developing a single cell assay using immunofluorescence microscopy to quantify adduct formation in sulfur mustard exposed skin (18). The results to date clearly show that antibodies can be developed against various chemical warfare agents or their specific adducts. These antibodies have been used to demonstrate that immunoassays capable of detecting chemical nerve agents in the micromolar (ppb) range and adducts in the nanomolar are quite feasible. Practically, immunoassays for chemical warfare nerve agents or vesicating agents have potential application in the emerging area of chemical weapons remediation or destruction. Workers required to verify the destruction of chemical weapons will need rapid semi-quantitative tests to determine that storage sites and surrounding areas have not been contaminated. There will also be a need for the same types of tests to screen workers for potential exposures to chemical weapons that would require medical treatment. The current antibodies do not have the requisite affinity for detecting nerve agents at a nanomolar concentration, which would be needed to determine levels of contamination, for determining potential exposure or to diagnose the type of exposure pursuant to making decisions on medical treatment. There are, however, no technical barriers to developing antibodies of higher affinity. Given that several reports of immunoassays for nerve agents have already been published, there is every reason to believe that future efforts will result in the development of immunoassays for nerve agents or their metabolites with the required sensitivity, selectivity and speed of response. In addition, detection of adducts of sulfur mustard at levels sensitive enough to confirm exposure to sub-vesicating dose are already available.
In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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LITERATURE CITED 1. Hammock, B . D., Gee, S. J., Cheung, P. Y . K . , Miyamoto, T., Goodrow, M. H . , Van Emon, J., and Seiber, J. N . in Pesticide Science and Biotechnology; Greenhalgh, R. and Roberts, T. R., Eds.; Blackwell Scientific Publications, Oxford, 1987; pp 309-316. 2. Brimfield, A . A., Lenz, D. E., Graham, C. and Hunter, Jr., K. W. J. Agr. Food Chem. 1985, 33, 1237-1242. 3. Hunter, Jr, K. W. and Lenz, D. E. Life Sci. 1982, 30, 355-361. 4. Al-Rubae, A. Y . The enzyme-linked immunosorbent assay, a new method for the analysis of pesticide residues. PhD Dissertations, The Pennsylvania State University, University Park, PA, 1978. 5. Van Emon, I., Seiber, I., and Hammock, B . Bull. Environ.Contam.Toxicol. 1987, 39, 490-497. 6. Heldman, E., Balan, A . , Horowitz, O., Ben-Zion, S., and Torten, M. FEBS Lett. 1985, 180, 243-248. 7. Hunter, Jr., K. W., Lenz, D. E., Brimfield, A . A., and Naylor J. A . FEBS Lett. 1982, 149, 147-151. 8. Lenz, D. E., Brimfield, A . A., Hunter, Jr., K . W., Benschop, H . P., de Jong, L . P. A., van Dijk, C., and Clow, T. R. Fund. Appl. Toxicol. 1984, 4, S156-S164. 9. Brimfield, A . A . , Hunter, K . W., Lenz, D. E., Benschop, H . P., van Dijk, C., and de Jong, L . P. A . Mol. Pharmacol. 1985, 28, 32-39. 10. Erhard, M . H . , Schmidt, P., Kuhlmann, R., and Losch, U . Arch. Toxicol. 1989, 63, 462-468. 11. Erhard, M . H . , Kuhlmann, R., Szinicz, L . , and Losch, U . Arch. Toxicol. 1990, 64, 580-585. 12. Rong, K.-T. and Zhang, L.-J. Pharmacology and Toxicology 1990, 67, 255259. 13. Grognet, J.-M., Ardouin, T., Istin, M . , Vandais, A . , Noel, J.-P., Rima, G., Satge, J., Pradel, C., Sentenac-Roumanou, H . , and Lion, C., (1993) Arch. Toxicol., 67, 66-71. 14. Dean, R. G. Defense Advanced Research Projects Agency, R & D Status Report, USA, 1982: AD-A122300. 15. Dean, R. G. Defense Advanced Research Projects Agency, R & D Status Report, USA, 1987: AD-A188149. 16. Zhou, Y . - X . , Yan, Q.- J., Ci, Y.- X . , Guo, Z.-Q., Rong, K.-T., Charg, W.B. and Zhao, Y.-F. Arch. Toxicol. 1995, 69, 644-648. 17. Lieske, C. N., Klopcic, R. S., Gross, C. L . , Clark, J. H . , Dolzine, T. W., Logan, T. P. and Meyer, H . G. Immuno. Let. 1992, 31, 117-122. 18. Cook, L . , Lieske, C. N. and Brimfield, A . A . Proceedings of the 1996 Medical Defense Bioscience Review; Baltimore, M D , 1996, pp 136-137. 19. Fidder, A . , Moes, W. H . , Scheffer, A . G., van der Schans, G. P., Baan, R. A., de Jong, L . P. A., Benschop, H . P.Chem.Res. Toxicol. 1994, 7, 199-204. 20. van der Schans, G . P., Scheffer, A . G . , Mars-Groenendijk, R. H., Fidder, A . , Benschop, H . P., Baan, R. A . Chem. Res. Toxicol 1994, 7, 408-413.
In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
