Geological Survey of Denmark and Greenland Bulletin
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Nr. 4, Review of Survey activities 2003, pp. 29-32 |
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29
Groundwater is the major source of drinking water in many
European countries, and in Denmark alone it accounts for
more than 99% of the drinking water supply. Within the past
decade pesticide residues have frequently been detected in
groundwater, in many cases at levels exceeding the 0.1 µg/l
limit set by the European Community. As a consequence,
drinking water abstraction wells have had to be closed in
many places in Denmark and other European countries, and
a vast amount of money is expended to monitor groundwa-
ter pesticide levels. A degradation product of the herbicide
dichlobenil, 2,6-dichlorobenzamide (BAM), is the most
common cause of drinking water well closure in Denmark.
Triazines and their metabolites also contaminate groundwa-
ter in many countries, and pose a similar risk to the drinking
water supply. Analysis of most pesticides and their degrada-
tion products is usually carried out by concentrating the sam-
ples by solvent extraction, and identifying the contaminants
using gas chromatography (GC) or high-pressure liquid
chromatography (HPLC) combined with mass spectrometry
(MS). These methods, although robust and well established,
are very time-consuming and require specialised instrumen-
tation. The large quantity of solvents used is another draw-
European countries, and in Denmark alone it accounts for
more than 99% of the drinking water supply. Within the past
decade pesticide residues have frequently been detected in
groundwater, in many cases at levels exceeding the 0.1 µg/l
limit set by the European Community. As a consequence,
drinking water abstraction wells have had to be closed in
many places in Denmark and other European countries, and
a vast amount of money is expended to monitor groundwa-
ter pesticide levels. A degradation product of the herbicide
dichlobenil, 2,6-dichlorobenzamide (BAM), is the most
common cause of drinking water well closure in Denmark.
Triazines and their metabolites also contaminate groundwa-
ter in many countries, and pose a similar risk to the drinking
water supply. Analysis of most pesticides and their degrada-
tion products is usually carried out by concentrating the sam-
ples by solvent extraction, and identifying the contaminants
using gas chromatography (GC) or high-pressure liquid
chromatography (HPLC) combined with mass spectrometry
(MS). These methods, although robust and well established,
are very time-consuming and require specialised instrumen-
tation. The large quantity of solvents used is another draw-
back to these methods, as the solvents themselves may be car-
cinogenic and are also well known contaminants of ground-
water. The development of cheap, more sensitive and more
rapid pesticide assays is therefore urgent.
cinogenic and are also well known contaminants of ground-
water. The development of cheap, more sensitive and more
rapid pesticide assays is therefore urgent.
Due to their very high sensitivity, immunological methods
have long been used in biological science for analysing a large
variety of organic structures, but have only recently been
introduced to environmental analysis. The benefit of such
assays is primarily their high sensitivity, which allows the
analysis to be undertaken without the need to concentrate
the samples, but also the facility of dealing with large num-
bers of samples. Compared to conventional analyses, im-
munological methods face two major drawbacks one
related to specificity and the other to the fact that only very
few chemicals can currently be analysed simultaneously. The
crux of the specificity problem is that although antibodies
react very specifically with particular chemical structures,
these same structures may be present in analogous com-
pounds. Thus antibodies developed to recognise, for example
the herbicide atrazine might also recognise other triazines
(Bruun et al. 2001). An important scientific challenge is
therefore the development of highly specific assays recognis-
variety of organic structures, but have only recently been
introduced to environmental analysis. The benefit of such
assays is primarily their high sensitivity, which allows the
analysis to be undertaken without the need to concentrate
the samples, but also the facility of dealing with large num-
bers of samples. Compared to conventional analyses, im-
munological methods face two major drawbacks one
related to specificity and the other to the fact that only very
few chemicals can currently be analysed simultaneously. The
crux of the specificity problem is that although antibodies
react very specifically with particular chemical structures,
these same structures may be present in analogous com-
pounds. Thus antibodies developed to recognise, for example
the herbicide atrazine might also recognise other triazines
(Bruun et al. 2001). An important scientific challenge is
therefore the development of highly specific assays recognis-
Immunological analysis of pesticides: a new tool
in groundwater testing
in groundwater testing
Jens Aamand, Leif Bruun and Claus Bo Vöge Christensen
Fig. 1. Development of monoclonal pesticide
antibodies is initiated by covalent conjugation
of the pesticide to a carrier protein. This
pesticide-carrier complex is injected into
mice and after approximately two months the
mice have produced antibodies against the
pesticide. Selected mice are sacrificed and
the spleen is removed to isolate the
antibody-producing cells. These cells are
difficult to cultivate in vitro, and they are
therefore fused with myeloma cells to ensure
the viability of the antibody-producing cells.
The fused hybridoma cells are cultivated,
tested and isolated to achieve monoclonal
cultures, which produce one type of
antibody only with special characteristics
such as binding efficiency and specificity.
Slightly modified from Aamand et al. (2003).
Geological Survey of Denmark and Greenland Bulletin 4, 2932 (2004) © GEUS, 2004
ing each individual compound, as well as assays recognising
groups of related chemicals.
groups of related chemicals.
With respect to the simultaneous analysis of numerous
chemicals, this can be resolved by implementing the new
biochip technology, which incorporates the parallellity of
sample screening. On a pesticide biochip many specific im-
munological assays are carried out in isolated small spots on
a glass or polymer surface. Each spot has a size of approxi-
mately 150 micrometers and forms a specific analysis. Such a
miniaturised platform will be usable for monitoring pro-
grammes where water samples have to be screened for a range
of chemical contaminants.
biochip technology, which incorporates the parallellity of
sample screening. On a pesticide biochip many specific im-
munological assays are carried out in isolated small spots on
a glass or polymer surface. Each spot has a size of approxi-
mately 150 micrometers and forms a specific analysis. Such a
miniaturised platform will be usable for monitoring pro-
grammes where water samples have to be screened for a range
of chemical contaminants.
The overall objectives of this study have been (1) to
develop immunoassays for high-sensitivity analysis of specific
pesticides and chemically related groups of pesticides, and (2)
to transfer the developed assays to a miniaturised biochip
platform in a manner allowing analysis of several pesticides
simultaneously.
pesticides and chemically related groups of pesticides, and (2)
to transfer the developed assays to a miniaturised biochip
platform in a manner allowing analysis of several pesticides
simultaneously.
Immunological analysis of pesticides
The basis for the development of new immunological analy-
ses is the antibody that reacts with complementary mole-
cules, the so-called antigens. Antibodies are part of the
immunological defence system in animals and humans for
protection against pathogenic vira and bacteria. Following an
ses is the antibody that reacts with complementary mole-
cules, the so-called antigens. Antibodies are part of the
immunological defence system in animals and humans for
protection against pathogenic vira and bacteria. Following an
infection, the organism produces antibodies that recognise
and bind to specific molecular structures on the surface of the
penetrating bacteria or virus (the antigens). Upon the bind-
ing of antibodies, other effector functions of the immune sys-
tem identify and destroy the bacteria. The chemical structures
of the pesticides themselves are too small to induce an im-
munological response. However, by linking the pesticides to
larger carrier molecules it is possible to deceive the immune
system into starting the production of antibodies against the
pesticide (Fig. 1).
and bind to specific molecular structures on the surface of the
penetrating bacteria or virus (the antigens). Upon the bind-
ing of antibodies, other effector functions of the immune sys-
tem identify and destroy the bacteria. The chemical structures
of the pesticides themselves are too small to induce an im-
munological response. However, by linking the pesticides to
larger carrier molecules it is possible to deceive the immune
system into starting the production of antibodies against the
pesticide (Fig. 1).
To initiate antibody production the pesticide-carrier com-
plex is injected into an animal, e.g. a rabbit or a mouse, thus
inducing an immunological response resulting in the pro-
duction of antibodies against the pesticide-carrier complex.
Antibodies are produced by so-called B-cells each producing
a single antibody species, which recognise a specific structure
on the pesticide. As the animal contains many B-cells which
all produce antibodies, a range of antibodies reacting with
different structures on the pesticide and with different affin-
ity will be generated. By purification of the antibodies from
the blood serum, a polyclonal antibody serum is obtained,
containing antibodies from different B-cells. However, it is
often more appropriate to produce monoclonal antibodies
(Mab), i.e. specific antibodies all arising from the same B-cell
clone.
inducing an immunological response resulting in the pro-
duction of antibodies against the pesticide-carrier complex.
Antibodies are produced by so-called B-cells each producing
a single antibody species, which recognise a specific structure
on the pesticide. As the animal contains many B-cells which
all produce antibodies, a range of antibodies reacting with
different structures on the pesticide and with different affin-
ity will be generated. By purification of the antibodies from
the blood serum, a polyclonal antibody serum is obtained,
containing antibodies from different B-cells. However, it is
often more appropriate to produce monoclonal antibodies
(Mab), i.e. specific antibodies all arising from the same B-cell
clone.
Production of monoclonal antibodies
When it has been established by serological screening that the
immunised animal produces antibodies with the correct
specificity, the spleen, which contains many antibody-pro-
ducing cells, is removed and grown in culture. Clones pro-
ducing antibodies with the desired properties are then
selected. It is possible to select antibodies that react with
chemical structures specific for a single pesticide molecule, or
alternatively a structure shared by a group of pesticides such
as the triazines.
immunised animal produces antibodies with the correct
specificity, the spleen, which contains many antibody-pro-
ducing cells, is removed and grown in culture. Clones pro-
ducing antibodies with the desired properties are then
selected. It is possible to select antibodies that react with
chemical structures specific for a single pesticide molecule, or
alternatively a structure shared by a group of pesticides such
as the triazines.
In addition to their high specificity monoclonal antibod-
ies also have the advantage of consistency. It is always possi-
ble to reculture the hybridomas and produce further an-
tibodies with exactly the same characteristics.
ble to reculture the hybridomas and produce further an-
tibodies with exactly the same characteristics.
Development of immunological assays
The next step following the selection of suited antibodies is
the development of an immunochemical pesticide assay. The
analysis is often carried out as a so-called competitive im-
munoassay in microtitre plates, which are preformed plastic
plates with 96 wells (Fig. 1). A known amount of pesticide is
immobilised on the bottom of the wells. The samples to be
analysed are added to the wells (typically 100 µl), followed by
the development of an immunochemical pesticide assay. The
analysis is often carried out as a so-called competitive im-
munoassay in microtitre plates, which are preformed plastic
plates with 96 wells (Fig. 1). A known amount of pesticide is
immobilised on the bottom of the wells. The samples to be
analysed are added to the wells (typically 100 µl), followed by
30
Fig. 2. Example of a competitive assay. In cases with high concentrations
of pesticides in the sample, fewer antibodies will bind to the pesticide
immobilised to the surface (e.g. the bottom of the microtitre plate) and
a low signal will be measured. If the samples do not contain the pesti-
cide, maximal amounts of antibody will bind to the surface resulting in
a high signal. Slightly modified from Aamand et al. (2003).
the addition of the antibodies. At this point the antibodies
can react with either the pesticide immobilised in the
microtitre plates or the pesticide in the sample itself. If the
concentration of the pesticide in the sample is low, more anti-
bodies will react with the immobilised pesticides and vice
versa. When the reaction is completed the microtitre plate
wells are washed, leaving only the immobilised pesticide-
antibody complex (Fig. 2). The antibodies can be directly
monitored if coupled with an enzyme, catalysing an enzyme-
substrate reaction that yields a coloured end product. The
accumulation of the end product is then monitored by
absorbance measurements and compared spectrophotometri-
cally to the absorbance of known standards (Fig. 3).
can react with either the pesticide immobilised in the
microtitre plates or the pesticide in the sample itself. If the
concentration of the pesticide in the sample is low, more anti-
bodies will react with the immobilised pesticides and vice
versa. When the reaction is completed the microtitre plate
wells are washed, leaving only the immobilised pesticide-
antibody complex (Fig. 2). The antibodies can be directly
monitored if coupled with an enzyme, catalysing an enzyme-
substrate reaction that yields a coloured end product. The
accumulation of the end product is then monitored by
absorbance measurements and compared spectrophotometri-
cally to the absorbance of known standards (Fig. 3).
New immunological assays have been developed for sev-
eral triazines including their degradation products (Bruun et
al. 2000a, b, 2001) and for BAM (Bruun et al. 2000c). All
assays have a very low detection limit in the range of
0.010.02 µg/l, making them ideal for monitoring specific
pesticide residues in ground- and drinking water.
al. 2000a, b, 2001) and for BAM (Bruun et al. 2000c). All
assays have a very low detection limit in the range of
0.010.02 µg/l, making them ideal for monitoring specific
pesticide residues in ground- and drinking water.
Analyses are typically carried out in four replicates, and
each microtitre plate also contains a number of pesticide
standards. A total of 13 samples can be analysed at each
microtitre plate within a period of 34 hours.
standards. A total of 13 samples can be analysed at each
microtitre plate within a period of 34 hours.
From microtitre plate to pesticide biochip
One of the drawbacks of the microtitre format is that it is
only possible to analyse for one pesticide in each routine.
However, changing the analysis format from microtitre plates
to biochips allows for the analysis of several compounds
simultaneously. The term `biochips' describes an analysis
where the chemical reactions are not separated by wells, but
are carried out on a planar surface such as a glass slide. The
principle of the analysis is the same as for the microtitre
plates, but the reagents are added as microspots (in the nano-
liter range) on the glass surface. Using a robot equipped with
printing pins, about 2000 samples/cm
only possible to analyse for one pesticide in each routine.
However, changing the analysis format from microtitre plates
to biochips allows for the analysis of several compounds
simultaneously. The term `biochips' describes an analysis
where the chemical reactions are not separated by wells, but
are carried out on a planar surface such as a glass slide. The
principle of the analysis is the same as for the microtitre
plates, but the reagents are added as microspots (in the nano-
liter range) on the glass surface. Using a robot equipped with
printing pins, about 2000 samples/cm
2
can be added as sep-
arate spots on the surface. All chemical reactions are then car-
ried out within the individual spots. For the pesticide biochip
fluorescence-conjugated monoclonal antibodies were used,
which enables the detection of separate signals from each spot
on the surface by use of a laser scanner (Fig. 4). As a result of
the small dimensions, the individual reactions equilibrate
faster and the complete analysis of a biochip can be carried
out within 90 minutes. The analyses on the biochips are also
more sensitive than on microtitre plates. We have developed
a pesticide biochip for BAM and atrazine with a sensitivity of
about 1 ng/l (Fig. 5), which is 100 times less than the limit
value for drinking water set by the EU (Belleville et al. 2003,
2004).
ried out within the individual spots. For the pesticide biochip
fluorescence-conjugated monoclonal antibodies were used,
which enables the detection of separate signals from each spot
on the surface by use of a laser scanner (Fig. 4). As a result of
the small dimensions, the individual reactions equilibrate
faster and the complete analysis of a biochip can be carried
out within 90 minutes. The analyses on the biochips are also
more sensitive than on microtitre plates. We have developed
a pesticide biochip for BAM and atrazine with a sensitivity of
about 1 ng/l (Fig. 5), which is 100 times less than the limit
value for drinking water set by the EU (Belleville et al. 2003,
2004).
Possibilities and limitations
The benefits of the immunochemical analyses compared to
chromatographic techniques are that: (1) less sample volume
is needed, which means an easier transport of samples to the
laboratory; (2) no solvents or other chemicals are necessary
which potentially could pollute the environment; and (3) the
immunochemical analyses are much cheaper to carry out.
chromatographic techniques are that: (1) less sample volume
is needed, which means an easier transport of samples to the
laboratory; (2) no solvents or other chemicals are necessary
which potentially could pollute the environment; and (3) the
immunochemical analyses are much cheaper to carry out.
At present, the immunochemical techniques only enable
the analysis of a few compounds simultaneously. In contrast,
chromatographic methods (e.g. HPLC or GC/MS) provide
the concentration of a range of compounds within the same
routine. However, use of the pesticide biochips opens the
possibility of analysis of more compounds simultaneously. At
present the biochip includes BAM and atrazine only, but in
theory it is possible to include additional pesticides as soon as
usable antibodies become available.
chromatographic methods (e.g. HPLC or GC/MS) provide
the concentration of a range of compounds within the same
routine. However, use of the pesticide biochips opens the
possibility of analysis of more compounds simultaneously. At
present the biochip includes BAM and atrazine only, but in
theory it is possible to include additional pesticides as soon as
usable antibodies become available.
Another problem to be faced is related to the specificity of
the antibodies. Many antibodies may react not only with the
targeted pesticide, but also with chemically related com-
pounds. This is the case for the atrazine antibodies that may
also react with other triazine herbicides (Bruun et al. 2001).
The specificity, however, is not a problem with BAM,
because the reactivity of the antibody with other compounds
is negligible (Bruun et al. 2000c).
targeted pesticide, but also with chemically related com-
pounds. This is the case for the atrazine antibodies that may
also react with other triazine herbicides (Bruun et al. 2001).
The specificity, however, is not a problem with BAM,
because the reactivity of the antibody with other compounds
is negligible (Bruun et al. 2000c).
The new pesticide biochip enables the analysis of pesti-
cides in a single drop of water in concentrations as low as 1 ng/l.
In principle the pesticide biochip allows the analysis of a
range of pesticides, but for the development of such multi-
component analysis further antibodies are needed with high
specificities to the individual pesticides.
In principle the pesticide biochip allows the analysis of a
range of pesticides, but for the development of such multi-
component analysis further antibodies are needed with high
specificities to the individual pesticides.
31
Fig. 3. Example of a standard curve. Note the inverse relationship
between pesticide concentration and signal. Slightly modified from
Aamand et al. (2003).
32
Acknowledgement
The present work is supported by the Immunalyse Project (Grant no.
9901188) financed by the Danish Research Agency.
9901188) financed by the Danish Research Agency.
References
Aamand, J., Bruun, L. & Christensen, C.B.V. 2003: Mus hjælper til med
pesticidanalyser. Dansk Kemi 84, 2931.
Belleville, E., Dufva, M., Aamand, J., Bruun, L. & Christensen, C.B.V.
2003: Quantitative assessment of factors affecting the sensitivity of a
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35, 10441051.
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Belleville, E., Dufva, M., Aamand, J., Bruun, L., Clausen, L. & Christen-
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clonal antibody for the sensitive detection of cyanazine and other s-
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Bruun, L., Koch, C., Jakobsen, M.H. & Aamand, J. 2000b: New mono-
clonal antibody for the sensitive detection of hydroxy-s-triazines in
water by enzyme-linked immunosorbent assay. Analytica Chimica
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Bruun, L., Koch, C., Pedersen, B., Jakobsen, M.H. & Aamand, J. 2000c: A
quantitative enzyme-linked immunoassay for the detection of 2,6-
dichlorobenzamide (BAM): a degradation product of the herbicide
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Bruun, L., Koch, C., Jakobsen, M.H., Pedersen, B., Christiansen, M. &
Aamand, J. 2001: Characterisation of monoclonal antibodies raised
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Fig. 4. Laserscan of a pesticide biochip designed to analyse for BAM and atrazine. Each spot represents a single analysis of a standard with a known
concentration. Within each concentration the six spots to the left are BAM and the six spots to the right are atrazine. Slightly modified from Aamand
et al. (2003).
Authors' addresses
J.Aa., Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: jeaa@geus.dk
L.B., Statens Serum Institut, Artillerivej 5, DK- 2300 Copenhagen S, Denmark.
C.B.V.C., Technical University of Denmark, Department of Micro- and Nanotechnology, Ørsted Plads 345, DK-2800 Kongens Lyngby,
Denmark.
J.Aa., Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: jeaa@geus.dk
L.B., Statens Serum Institut, Artillerivej 5, DK- 2300 Copenhagen S, Denmark.
C.B.V.C., Technical University of Denmark, Department of Micro- and Nanotechnology, Ørsted Plads 345, DK-2800 Kongens Lyngby,
Denmark.
