Immunochemical methods for environmental monitoring

Nucl.

Pergamon

0969-8051(94)E0032-E

lmmunochemical

Med.

Biol.

Vol. 21, No. 3, pp. 557-572,

1994

Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0969-8051194 $6.00 + 0.00

methods for environmental

monitoring

Gianfranco GIRAUDI and Claudio BAGGIANI Dipartimento di Chimica Analitica, Universita di Torino, Torino, ltaly

ABSTRACT

lmmunochemical

methods for environmental

analysis

must be taken into consideration

for their ability to expand the potential of analytical measures rather than for substituting methodologies.

current

Moreover, the full potential of these methods has yet to be realized. Indeed, the

terms and concepts of immunology

are new to most analytical chemists, even if environmental

science has always been an interdisciplinary of immunoassays

field. On the other hand, the clinical development

means that much experience

urine, and tissue samples. The immunochemical waste chemicals,

more

poses new challenges

has been gained in the analysis

analysis of samples from soils, ground water,

in sample preparation that have yet to be extensively

studied, and in the future there may be immunoassays associated with environmental

of blood,

better suited for the particular

problems

monitoring.

All correspondence should be addressed to: G. Giraudi, Dipartimento Universita’ di Torino, Via P. Giuria 5.10125 Torino, ITALY. 557

di Chimica Analitica,

GIANFRANCO GIRAUDIand CLALJDIOBAGGIANI

558

The advantages of immunochemicai methods are well known to those working in the clinical field. These methods offer great potential for rapid screening and quantative analysis of virtually all classes of compounds for which specific antibodies can be obtained, so it is not surprising that immunochemical methods have been developed

for measuring environmental

contaminants.

The application

of

immunochemical techniques to environmental problems has been extensively reviewed (Vanderlaan, 1988 and Van Emon, 1992). Driving forces for the development of these methods are mainly rising costs, increasingly loads of samples, and the length of time required for classical trace analysis. Indeed, in all countries there is an increasing demand for the identification of the source and extent of contamination by chemical pollutants and’for the distribution of pesticides and toxic chemicals in food and environment. This situation leads to analyse an increasing number of samples, which however are often inadequate to ensure detection of pollution. Moreover, there is a too long a delay between sample collection and communication of results back to the site. All these problems stem from the cost, sophistication, and time involved in multiresidue analytical chemistry. Even with the best analytical methods, problems arise when research laboratory procedure are scaled up to handle thousands of samples under mass screening conditions. There is also an increasing need for rapid and simple tests that can be performed on the site without transfer to an analytical laboratory. These field methods can be used by personnel unfamiliar with analytical chemistry methodologies. All these problems motivate the search for rapid, low-cost residue detection immunochemical methods.

methods, such as

Euro-Immunoanalyse

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559

The association between environmental pollutants and immunochemistry is not recent. The first work on this subject could be considered the generation of antibodies to dinitrophenol (Farah, 1960). The immune response to this molecule has been extensively investigated to study the regulation of the immune system. The environmental interest lies in the fact that dinitrophenol is, after all, not only a convenient chemical for studying immunology but also a priority pollutant for the Environmental Protection Agency of USA (Sittig, 1980). However, only in the last two years a growing number of publications, meetings, and a proliferation of companies marketing immunoassays for environmental and food residue analysis have been observed (figure 1).

1981

1982

1983

1984

1985

1988

1987

1988

1989

1990

1991

1992

Figure 1 - Number of papers on immunochemical methods for environmental analysis, published in the last twelve years (data for 1992 refer to first six-month period only).

The main applications of immunochemical methods for environmental analysis are immunoassays, commonly based on the ELISA technique, for the detection of a variety of contaminants in water, air, soil, food. For example, specific assays have been developed for many pesticides, industrial chemicals, mutagens, carcinogens, protein products from microorganisms, and various biomarkers of exposure such as DNA adducts (table 1). Most of the methods reported have detection limits in the range of a few ppb (or nglml). DNA adducts can be detected with a sensitivity greater than all the other compounds.

560

GIANFRANCOGIRAUDIand CLALJDIO BAGGIANI

Table 1 - lmmunochemical

methods reported in the literature for environmental

Compound

Immunoassay format

Pesticides Alachlor Aldicarb Atrazine Bentazon Benomyl Chlorsulfuron 2,4-D and 2,4,5-T Diclofop-methyl Diflurbenzuron Endosulfan Fenpropimorph lprodione Metalaxyi Molinate Monolinuron Diuron Norflurazon Norflurazon desmethyl Paraquat Parathion Permethrin Picloram Terbutryn Triadimefon Triazine herbicides

ELISA ELISA ELISA ELISA ELISA RIA ELISA RIA EIA, FIA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA RIA ELISA ELISA ELISA ELISA ELISA

Mutagens, carcinogens, toxic chemicals 4Aminobiphenyl RIA metabolite Aminoimidazoazaarenes ELISA Benzene ELISA toluene, xylene Benzidine metabolite RtA ELISA Benzo[a]pyrene Diethylstilbestrol RIA Nitroaromatics ELISA Nitrofluoranthrenes RIA PCBs RIA RIA ELISA Pentachlorophenol (PCP) ELISA PCDDs and PCDFs ELISA RIA ELISA 2,3,7,8-TCDD RIA

Detection limit

analysis.

I50 value @g/ml)

1 wb

0.75 ppm 0.32 2 ppb 0.35 ppm

1OOPg 1 wb 3 ng/ml 13 nglml

2 90 7 70 3.9

3.0 0.5 20 ppb 14-22 nglg 0.1 us/g 1 ng/ml 10 nglml 0.1-l nglml

3.0 50 17

10 nglml 0.5 ppm 0. l-l 0 nglml

250 2.4

2.5 1.0 5 ppm 0.25 500 0.1 1 ng/ml 120 2-16 ppb 1 50 ppm 3 ppb (field assay) 1 ppb

0.7 pmol

1 1

561

Euro-Immunoanalyse ‘93

Table 1 (continued) Microbial toxins Aflatoxin Bl Aflatoxins Mycotoxins

0.1 ng/ml

ELISA RIA RIA ELISA ELISA ELISA

Ochratoxin T2-toxin DNA Adducts Aflatoxin Bl-DNA 1-aminopyrene-DNA Benzo[a]pyrene-DNA Ethylated and methylated bases

3.3 0.1 nglml 0.1 ng/ml 1 nglml 10 nglml

ELISA ELISA ELISA

6.0 0.05 0.05

RIA

0.02

a RIA = Radioimmunoassay; FIA = Fluorescence Immunoassay; ELISA = Enzyme Linked lmmunosorbent Assay; EIA = Enzyme Immunoassay. b List adapted mainly from Vanderlaan (1988) and Van Emon (1992).

While in the clinical field the most frequent use of immunoassay is to identify macromolecules, in the environmental field target analytes are small organic molecules (i.e., haptens). Typical steps required to develop an assay for a chemical pollutant are listed in figure 2. The first important1 point is the right choice of the target analyte. The pollutant itself could be the target if it displays a good stability in the environment or, in the opposite case, a relevant degradation product. Alternatively, both pollutant and degration products could be usefully assayed to monitor the overall pollution caused by the chemical. One must plan the useful hapten by defining the type and position of the spacer arm, then synthesize it. The subsequent step is obviously the conjugation to a carrier protein to obtain the immunogen, by hapten activation or by modification and activation of the protein. The immune response is then induced by injecting the hapten-oarrier complex into an animal to obtain antibodies. When polyclonal or monoolonal antibodies are available, assay development and optimization must take into account all experimental factors known to affect assay response (i.e. antibody concentration, coating antigen concentration, combination of coating-antigen concentration, pH and ionic strength of the reaction mixture). Moreover, solvent tolerance of the assay needs to be evaluated, because most environmental pollutants are directly assayed after solvent extraction from solid samples (e.g.

soil)

and/or

the

assay

must

be

performed

in

a

reaction

mixture

containing

GIANFRANCOGIRAUDIand CLAUDIOBAGGIANI

562

Target analyte

I

Hapten planning and synthesis Position and type of spacer arm must be defined 1

Conjugation

to carriers

- Modify protein - Activate modified protein - Conjugate with target compound or its analogue

- Activate hapten - Conjugate with carrier protein

1

Immunization Either antisera or monoclonal antibody 1

Assay development

and optimization

- Antibody concentration - Coating antigen concentration - Combinationof coating-antigen concentration -PH - Ionic strength - Solvent tolerance - Cross-reactivity I

Laboratory

method

precision, detection limit

I

9

Field-portable L

method

speed of analysis

Figure 2 - Typical steps in the development of an immunoassay for environmental pollutants.

a percentage of solvent required to overcome the limited solubility of the analyte itself. Assay optimization can be directed to the development of a classical laboratory method, to obtain good precision and a low detection limit, or to the development of a field-portable method characterized by speed of analysis and very simple protocol.

The critical step is dearly hapten planning and synthesis. This is dependent on the criteria chosen to develop the assay (figure 3). Hapten planning, position and type of the spacer arm can be defined either to elicit antibodies specific for the target analyte or antibodies selective for a class of compounds of similar chemical structure. If one is able to obtain specific antibodies, one can develop a quantitative

Euro-lmmunoanalyse

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immunoassay that can be used as an alternative to conventional chemical analysis. On the other hand, by means of antibodies with a broad reactivity, one can perform a screening assay on several environmental samples. Subsequently, positive samples can be subjected to multiresidue analysis by conventional analytical techniques, such as HPLC or Gas Chromatography.

Choice of the structure Hapten synthesis lmmunogen I Specific antibodies to analyte

Selective antibodies (class specificity)

I

* / Quantitative 7

]

I

r--1

ScrFng

assay

I

alternative to conventional techniques

positive samples subjected to conventional analysis *

Immunoassay

Figure 3 - Criteria to develop immunoassays for environmental analysis

These different strategies can be exemplified by two research projects we are developing. The first is related to the “Antarctica Project”, supported by the Italian National Research Council (CNR). Search and quantitative determination of trace pollutants in the Antarctica environment is one of the aims

564

GIANFRANCO GIRAUDI and CLAUDIOBAGGIANI

of this project. In this field, one of our goals is to develop possible

pollution

insecticide,

deriving

extensively

from

isomers

of aromatic

trichloromethyl

chlorine,

immunoassays

4,4’dichlorodiphenyI-2,2,2_trichloroethane

used in South America.

analysis of DDT by immunoassay

quantitative

(DDT),

for monitoring a well

The chemical structure of DDT is shown in figure 4. The

requires highly specific antibodies able to discriminate by-products

known

of industrial

synthesis

among position

and photodegradation

products

of

moiety.

In order to prepare an immunogen,

a strategy is now been developed

for an a priori evaluation

suitability of several haptens (figure 4) to elicit antibodies of a desired specificity. A comparison the tridimensional

the

between

structure of these haptens and that of DDT shows that the structure of the DDT analog

with a carboxymethyloximo

arm (4,4’dichlorobenzophenon-O-(carboxymethyl)oxime,

DDT-CMO)

is very

different from the target analyte because of the orientation of aromatic rings. The situation appears more favourable

in the case

dichlorodiphenyl-2-ethanol trichloromethyl

moiety

a hemisuccinyl

ester

hemisuccinate,

DDT-HS)

(4,4’dichlorodiphenyl-1

derivative

without

the trichloromethyl

or with a hemisuccinylamide

-amino-2,2,2-trichloroethane

moiety

(4,4’-

analog, which retain the hemiglutarimidate,

DDT-

EGm). To evaluate electronic

properties

photodegradation software

what derivative

and several

products, commercial

(Autodesk‘s

mechanical

of DDT

could be used as a good immunogen,

HYPERCHEM

analogs)

molecule with DDT, two indexes were considered.

(by-products

of industrial

of the geometrical

synthesis,

and the molecular

structure corresponding

with the MM+ force field method. To compare

to a

a given

From the minimum energy structure the distance (d) of

invariant for all the molecules examined),

were calculated.

Then the

value for the DDT

atom by atom. The mean value for each molecule was taken as an index (named differences

and

with the AM1 quantum

quadratic difference between these values for a given molecule and the corresponding were calculated,

shape

employing specific computer

density (q) was calculated

molecule,

energy was calculated

all atoms from the central one (Cl,

molecules

(table 2) were calculated

2.0). Electron

method on a geometry-optimized

minimum conformational

related

the structural

with DDT. Similarly, quadratic

differences

of electronic

Adz)

density between

each molecule and DDT were calculated atom by atom, and the mean value for each molecule was taken as an index (named Aq2) of the electronic properties, A plot of Aq2 vs. Ad2 (figure 5) makes it possible to evaluate in what measure a given molecule differs from DDT by considering

its distance from the origin

of the plot, From this plot, a DDT derivative suitable to elicrt antibodies with good specificity seems to be the hemisuccinyl ester (DDT-ES). serum albumin.

Therefore,

an immunogen was prepared by coupling DDT-ES to bovine

565

Euro-Immunoanalyse'93 4,4'-DICHLOROBENZOPHENON -0--OXItlE

4,4-DICHLORODIPHENYL-2_2_2-TRICHLOROETHANE

0-carboxymethyl side-arm

trichloromethyl moiety

-LkICHLORODIPHENYL-2-ETHANOL SUCCINATE

thy1 :cinate !-arm

Figure 4 - Chemical structure of DDT and some related derivatives prepare immunogens.

suitable to

GIANFRANCO GIRAUDI and CLAUDIO BAGGIANI

566

2

3. z

0.1 g*

E Na"

8'

l 4

5. 7.

13 0.01 :

r-'-~-~---*-.--‘- ib I

12

rv14

6'

v

7

15' 0.001 -

Ad2, mean 0.007

o,o-DDT 0

0.005

0 o.m-DDT

I m,p-DDT.

0.003

o,p-DDT

0

m,m-DDT

DDT-EA DDT-ES

0

0.001 0.01

0.02

0.03

0.04

0.05

0.06

Figure 5 - Plot of the mean differences in the geometric and electronic properties of several DDT analogues. Numbers refer to the compounds listed in Table 2.

Euro-lmmunoanalyse

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Table 2 - List of molecules related to DDT and taken into consideration for the strategy to develop specific anti-DDT antibodies.

By-products of industrial synthesis 1 - 4,4’dichlorodiphenyl-2,2dichloroethylene 2 - 2,4’dichlorodiphenyI-2,2dichloroethylene 3 - 4,4’dichlorodiphenylsulphone 4 - 2chlorophenyl-2,2,2-trichloroethyl-(4chloroethyl)-sulphonate 5 - 1chloro-(2chlorophenyl)-acetamide 6 - 4-chlorophenyl-2,2,2-trichloroethanol 7 - 1-chloro-(4-chlorophenyl)-acetamide 8 - 4chlorobenzenesulphonic acid Photodegradation products 9 - cis/trans-2,4,4’-trichlorodiphenyl-2,2dichloroethylene 10 - 4,4’dichlorodiphenyI-2chloroethylene 11 - 369, IO-tetrachlorophenantrene Commercial analogs 12 - 4,4’-dichlorodiphenyl-2-ethanol 13 - 4,4’-dichlorodiphenyl-1 -acetamido-2,2,2_trichloroethane 14 - 4,4’dichlorobenzophenone 15 - 4,4’-dimethoxydiphenyl-2,2,2-trichloroethane Derivatives suitable as immunogens a - 4,4’dichlorobenzophenone-O-(carboxymethyl)oxime b - derivatives of 4,4’dichlorodiphenyl-2,2,2-trichloroethyl-l-amine: hemisuccinamide, hemiadipamide and carboxymethylamine DDT-ES: 4,4’dichlorodiphenyl-2sthanol henisuccinate DDT-EA: 4’4’-dichlorodiphenyl-2-ethanol hemiadipate

The second example that can elucidate the different strategies for developing immunochemical methods for environmental analysis, is given by a research we are undertaking to monitor aromatic hydrocarbons (AHs) - such as benzene, toluene and xylene - in industrial and urban areas. In this case, antibodies to AHs should not discriminate between aromatic molecules of the same group, such as benzene and toluene, but between hydrocarbons and other classes of compounds of similar structure (phenols, nitro- and halo-derivatives, pyridines, ...). By applying the same quantitative criteria of evaluation used for DDT, phenylbutyric acid and phenoxypropionic acid were coupled to bovine serum albumin to obtain two immunogens that should have a different selectivity towards various aromatic molecules.

GIANFRANCOGIRAUDI and CLAUDIO BAGGIANI

568

immunizations antibodies

to elicit both polyclonal

are obtained,

and monoclonal

it will be possible

to verify

antibodies

are in progress and when

if the quantitative

criteria

used to planning

immunogens can be applied successfully.

Currently, pollutants

all analytical

are based

methods

on instrumental

normally without interferences

applied

in the environmental

techniques

(mostly

documented

chromatographic),

well characterized

established

for environmental

for other analytical techniques

(O’Rangers,

contaminants

of and

must fulfill

1990). They must be well

and a report on their application should illustrate clearly the detection limit of the assay, need

for use of positive and negative controls, effect of various interfering substances, limits of applicability to various environmental environmental

interferences

all these

aspects

must be documented

from crude sample preparation.

an immunoassay

immunoassay

considered,

to the reliability immunoassays

immunoassays

criterion to evaluate

analytical techniques,

require

may be affected

by

the reliability of

such as HPLC, GC or GUMS.

on these aspects and on possible problems associated

with the particular

allows the analyst to evaluate the method correctly and to become confident as

of the results

to environmental

should be considered.

Because

as performance

Another fundamental

is a comparison with conventional

documentation

if any, matrix effect and

matrices, the comparison of calibration curves in buffer and

matrix which often is added with solvents or detergents.

little or no cleanup,

A complete

a variety

from matrix effects, due to an extensive cleaning up before the analytical

step. To be classed with the above methods, all immunoassays the same requirements

field to determine

obtained.

Finally,

among

the criteria

to be evaluated

for applying

analysis, the cost of the assay compared with that of current techniques

From this point of view, the limited cost associated

with immunoassay

is highy

favourable when analyzing a great number of samples. However,

despite the fact that immunochemical

analysis and rapid screening obtained,

offer a great potential

of virtually all classes of compounds

in the environmental

the field and immunochemical

methods

analytical

for quantitative

for which specific antibodies

laboratory the conventional

can be

methods of analysis still dominate

methods are generally not taken into consideration.

Among the reasons for

this limited use is the length of time needed for development

of the method, which is typically six to eight

months after development

Moreover,

of the appropriate

antibodies.

there

are important

regulatory

barriers, because the regulatory agencies may reject the methods if it does not meet certain acceptance criteria,

such as tests on real-world

conventional

samples

and/or

correlation

analysis. Another important reason why immunochemical

into environmental

laboratories

is because

the term and concept

and comparability

of results

with

methods have not made their way of immunochemical

methods

are

Euro-lmmunoanalyse

unfamiliar to most analytical sound quantitative environmental

because

chemists, which consider these assays as bioassays

analytical

methods.

There

is a need to validate

chemists can become familiar and appreciative

Despite

the positive qualities

of immunoassays,

any one antibody may permit detection

residues,

one must decide in advance

antibody. Although multiresidue

the immunization

compound. analytical

they do have certain

what compound

Each compound

is to be measured

For example,

similar cross-reacting

a cocktail of antibodies,

the synthesis

to expect in a sample,

a conventional

for each new multiresidue

On the other hand, if the problem is one of screening then immunoassay

load is high enough,

because

immunoassays

the

of its own hapten-protein

a large

may be the more appropriate

technology. Even if good methods already exist for a compound, immunoassays if the sample

limitations.

of its own animals, etc., and the process must be repeated

is still preferable.

so that

and select an appropriate

by assembling

requires

number of samples for a limited number of compounds,

choose

immunoassays

of a limited set of structurally

If one does not know what chemicals approach

several

rather than chemically

of the potential of the technique.

analysis can be performed

assay must be built up gradually. conjugate,

569

‘93

may still be the method to

are suitable for parallel

sample

processing and automation. On the other hand, the immunochemical methodologies

methods for a given analyte apply well when traditional

require a very complex sample treatment, are not adequately

of efficient preconcentration

methods. In this respect, immunochemical

sensitive and there is a lack

methods can be perhaps unique

analytical methods for analysing biological pesticides.

Other applicationsof immunochemicalmethods

One of the most powerful applications This

unique

immobilized

separation analytes.

technique However,

of immunochemical

has been

used

in the application

mainly

methods is the immunoaffinity techniques. for isolating

to environmental

specific

problems,

of small molecules

(Reum, 1981 and Sharman,

of

in the isolation and

1991). A typical analytical

immunoaffinity technique is reported in figure 6. After the analyte is solvent-extracted

using

the immobilization

antibodies on to stationary supports makes it possible to obtain a great improvement separation

antibodies

scheme using the

from the sample, the

solution containing the specific analyte is passed through a column packed with the antibody-coated

GIANFRANCOGIRAUDIand CLAUDIOBACGIANI

570

SAMPLE

EXTRACTION

-

AFFINITY CHROMATOGRAPHY (immobilized Ab)

rl

IMMUNOASSAY

Figure 6 - Typical scheme of immunoaffinity techniques applied to environmental samples.

support (acqueous samples can often be applied directly to the column). A solvent system that is compatible with the antibody yet still idrophobic enough to extract compounds of interest from matrices such as soil or oils and lipids must be added. During this step, the immobilized specific antibody captures the target analyte, separating it from other components in the solution. The analyte can then be removed from the column by dissociating the antibody-analyte complex with a proper solvent. Elution with strong salts, pH adjustment, or elution with organic solvents is the most commonly used method. If the analyte is analyzed by instrumental techniques (HPLC, GC or GUMS),

the eluting system is usually the same

solvent required by the cromatographic step. Alternatively, the analyte could be eluted with a solvent compatible with the immunoassay and analyzed by that method. lmmunoaffinity techniques can be applied not only to specifically remove the analyte of interest from interfering substances, but can be a powerful prewncentration

technique when no other satisfactory method exists. The advantage of the

immunoaffinity technique in sample treatment before analysis relies on the efficiency of separation and the low solvent consumption due to the reduced chromatographic steps compared to a classical cleanup procedure, with consequent reduction of cost of the analysis, often associated with sample preparation. Moreover, the same antibody used in an immunoassay can be used to develop immunoaffinity techniques.

Euro-Immunoanalyse

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571

Other areas in which immunochemical methods can be applied are specific sensors based on the analyte-antibody

interaction, such as antibody-based biosensors (Hall, 1991) and personal exposure

monitors. In a biosensor (a device containing a biological component coupled with a transducer) same of the properties of the transducer are altered and measured whenever an interaction occurs between the target analyte and the biological component of the sensor. A device of this type, base on an antibody immobilized on a fiber optic probe, has been described and applied to remote measurements (Alarie, 1990). Personal exposure monitors are based on the principle of immunoaffinity and use a specific antibody to capture volatile compounds directly from ambient air for the direct measurement of vapors. Another design system uses microdialysis tubes with specific antibodies immobilized inside, and the membrane acts as an air-to-liquid interface, allowing volatile compounds to diffuse into the aqueous medium and to become bound to immobilized antibodies (Drinkwine, 1991). Finally, an ELISA method for the analysis of mercuric ions in water has been reported (Wylie, 1991). However, although the availability of immunoassays to detect organometallic compounds or inorganic ions would be very useful for monitoring drinking water sources, the development of such methods requires further studies based on metal chelating systems and host-guest complexation, because so far no general methods to obtain antibodies to metallic ions has been reported. Future applications of immunochemical techniques could be based on catalytic antibodies for the preparation of environmental samples for analysis, e.g., to catalyze a particular cleanup or derivatization procedure or to detoxify an hazardous waste.

Conclusions

lmmunochemical methods for environmental analysis must be taken into consideration more for their ability to expand the potential of analytical measures rather than for substituting current metodologies. Moreover, the full potential of these methods has yet to be realized. Indeed, the terms and concepts of immunology are new to most analytical chemists, even if environmental science has always been an interdisciplinary field. On the other hand, the clinical development of immunoassays means that much experience has been gained in the analysis of blood, urine, and tissue samples. The immunochemical analysis of samples from soils, ground water, waste chemicals, poses new challenges

in sample

GIANFRANCO GIRAUDI and CLAUDIO BAGGIANI

572

preparation

that have yet to be extensively

suited for the particular problems associated

studied, and in the future there may be immunoassays with environmental

better

monitoring.

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Alarie J. P., Bowyer J. R., Sepaniak

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Farah F. S., Kern M. and Eisen H. N. (1980) J. Exp. Med. 112, 1195. Hall E. A H. (1991) Biosensors, O’Rangers

J. J. (1990)

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F. W. and Schuster S. M. (1991) Anal. &o&em.

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