Chapter 3 Monoclonal
Antibody Technology
Program
Stephen Krogsrud and Kenneth T. Lang
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U.S. Army Toxic and Hazardous Materials Agency, Aberdeen Proving Ground, MD 21010-5401
The U.S. Army Toxic and Hazardous Materials Agency (USATHAMA) has sponsored the development of methods for analysis of tetryl, dieldrin, benzene, and p-chlorophenylmethylsulfone using monoclonal antibodies. While the work with tetryl has resulted in a test with a detection limit of approximately 2 ppm, work is s t i l l in progress to develop methods for the other three analytes. The U.S. Army Toxic and Hazardous Materials Agency/ or USATHAMA., is a Field Operating Agency of the U.S. Army Corps of Engineers that offers a wide spectrum of environmental support to Army installations nationwide. Services include œnducting remedial investigations and feasibility studies, as well as research into new methods of waste minimization, remediation, and environmental analysis. USATHAMA provides these services through contracts with environmental engineering firms throughout the country. Research projects, as well as routine analysis, are performed by contract laboratories, since USATHAMA has no laboratory facilities of its own. Currently, USATHAMA is investigating or cleaning up environmental problems at over 83 installations. These include depots and equipment rebuild facilities, ammunition plants, and installations listed in the cxxigressionally mandated base closure plan. In this work a wide variety of cmtaminates have been encountered from sources such as plating sludges, degreasers, paint and solvent wastes, and fuels/lubricants. Standard U.S. Environmental Protection Agency methods are generally used by USATHAMA to analyze environmental samples, but these methods are not available for a l l compounds of interest. Also, i t is often desirable to have a method of analysis which has
This chapter not subject to U.S. copyright Published 1990 American Chemical Society In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS
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22
a higher sample throughput than standard laboratory methods or which can be done quickly i n the f i e l d . Analyses using monoclonal antibodies can f i l l these needs, as well as offering the possibility of high selectivity, low detection limits, and low cost per sample. As USATHAMA i s not currently using immunoassay techniques i n any of i t s projects, the decision was made to develop, as a t r i a l , methods for four carpounds which are of concern at various installations. These compounds are: tetryl (trinitrophenyliie^ benzene, dieldrin, and p-cMorophenylnethylsulfone. These cxxtçounds are shown i n Figure 1. The goal i s to produce a test for water samples which can be adapted to either lab or f i e l d use and can, with minimal sample preparation, measure target analytes i n the low ppb range. The tetryl work has been completed. Work continues at the present tine on the development of analyses for the remaining compounds. The work i s being performed by the organizations listed in the acknowledgements. Approach The problem of developing an immunoassay i s basically one of isolating a monoclonal antibody with the required reactivity and specificity. These antibodies are produced by an animal's immune system i n response to inoculation with an antigen. In the work reported here, the animals used were mice (Balb/c) or rabbits (New Zealand White). The antigen was, of course, different for the development of each antibody. As the animal's immune system w i l l not recognize a compound with a molecular weight as low as that of the compounds i n Figure 1, i t was necessary to prepare a hapten-protein conjugate, or immunogen. The haptens synthesized are shown i n Figure 2, along with the proteins to which they were conjugated. In general, i t was desired to bind the hapten to the protein i n such a way that the most "distinctive " portion of the hapten was exposed. Analogs of each target analyte were synthesized containing an acid group at the desired point of conjugation with the protein. This can be seen i n Figure 2. It was realized that benzene, which lacks distinctive functionality, would provide the greatest challenge to finding a specific antibody. Results Although i n most cases only one immunogen was prepared, for dieldrin three immunogens were prepared. A study was made of the three to determine which had the greatest hapten concentration. The study showed hapten : protein molar ratios of 8.5, 25, and 71 for the BSA, OVA and THY proteins, respectively. These ratios were obtained by infrared (IR) analysis as well as elemental chlorine analysis. Chlorine analysis was used as only the dieldrin molecule contains covalently bound chlorine. The conjugates with the highest hapten loadings, OVA and THY, were used for immunization i n the dieldrin case.
In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
KROGSRUD AND LANG
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Tetryl
N0
Monoclonal Antibody Technology Program
Benzene
2
ρ - chlorophenvlmethvlsulfone
Dieldrin
CI
CI
ο Figure 1. Oaiçounds Selected for Immunoassay Development.
In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
In Immunochemical Methods for Environmental Analysis; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
Benzene Immunogen :
Figure 2.
Ρ - Chlorophenylmethyleulfone Immunogen :
Tetryl Immunogen :
Dieldrin Immunogen :
Inraunogens Used i n Each Project.
COOH
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(BSA)
(BSA)
(BSA)
3.
KROGSRUD AND LANG
Monoclonal Antibody Technology Program
After a series of inmunizations with the appropriate immunogen, lymphocytes were collected and fused to produce hybridomas. These were screened for antibodies with a f f i n i t y for the desired analyte. A summary of the results i s given i n Table I. Table I. Hybridoma Production
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Analvte Tetryl Dieldrin Benzene p-Chlorophenylmethylsulfone
Number of Fusions
Hybridomas Isolated With Positive Antibodies
2 5 8
16 8 0
9
1
As previously stated, cloning and antibody testing i s complete for the tetryl analysis at this time, however work continues with the other three analytes. The lack of functionality undoubtedly has contributed to the difficulty of producing an antibody for benzene. In the tetryl case, the most sensitive antibody was selected and used i n an assay based on competitive inhibition. The detection limit i n water of the method i s approximately 2 ppm. The cross-reactivity of the antibody i s given i n Table II.
Table II. Tetryl Monoclonal Antibody Cross-Reactivity Compound Aniline 2,6 -Dinitroaniline 2,4-Dinitroanaline 1,3-Dinitrobenzene 1,3,5-Txinitrobenzene 2,4-Dinitrotoluene 2,6-Dinitrotoluene 2,4,6-Txinitrotoluene N-Methyl-2-Nitooaniline N-Met±yl-4-Nitroaniline
Reactivity 2 1 1 10 5 < 1 2 2 < 1 < 1 <
