Chapter 3
Rapid On-Site Immunoassay Systems Agricultural and Environmental Applications
J. H. Rittenburg, G. D. Grothaus, D. A. Fitzpatrick, and R. K. Lankow Agri-Diagnostics Associates, 2611 Branch Pike, Cinnaminson, NJ 08077
A simple immunoassay system has been developed for rapid on-site analysis and quantitation of chemical residues. The system consists of an assay device with an absorbant core to which antibody can be immobilized, and a small handheld reflectometer for quantitation of the assay results. The competitive immunoassay is performed by adding reagents to the surface of the device from dropper bottles. As each solution is absorbed into the device, it passes through the surface zone of immobilized antibody allowing the antibody -antigen reactions to proceed. The immunoassay can be completed within 10 minutes with a visually observable color endpoint. Each assay device contains a negative control reference zone that is used for comparison to the sample zone. Results are quantitated using a handheld, dual beam, reflectometer that compares the color intensity of the sample zone to that of the reference zone. The development of both a multiwell and field usable immunoassay for quantitation of alachlor in the low to sub ppb range is described. The accurate and precise analysis of pesticides is a critical requirement for the registration and use of pesticides throughout the world. Parent molecules, key metabolites and chemical breakdown products must be identified and studied in well designed laboratory andfieldresearch trials. Environmentally sound management practices rely on significant amounts of information about the levels and movements of pests, pathogens and specific chemical treatments within the environment. The methods available for such analysis have become extremely sophisticated and sensitive in response to the need to detect lower and lower levels of contaminants in crops, water, soil, and farm animals. Despite the tremendous sophistication of pesticide residue and environmental chemical analysis, there remain a number of serious limitations to certain aspects of classical analysis. A number of those limitations can be addressed through the application of immunoassay technology to residue analysis (L4). Immunoassays rely on highly specific antibody proteins and relatively simple analytical apparatus to detect and quantify a wide variety of target materials in a broad range of analytical matrices. Since the reagents are specific, immunoassays can generally be performed with relatively crude sample preparations. Reduced sample preparation, simple 0097-6156/91/0451-0028$06.00/0 © 1991 American Chemical Society
3.
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assay procedures, high throughput capabilities, and relatively inexpensive automation make immunoassay procedures much less expensive on a per sample basis than conventional methods. Additionally, immunoassays can be readily adapted to simple and rapid on-site testing methods that generate timely information enabling better informed decisions to be made. Immunoassay Technology Immunoassays are analytical techniques based on the specific, high affinity binding of inducible animal-derived proteins called antibodies with particular target molecules called antigens. Antibody formation by higher vertebrates is remarkable both i n the specificity of the induced antibody to its target and in the variety of organic molecules and macromolecules that are able to induce a specific antibody response. The primary binding between the antibody and the target antigen forms the basis of the immunoassay, and a wide variety of immunoassay "formats" have been developed to allow either visual or instrumental measurement o f this primary binding reaction. The tremendous variety of immunoassay formats and reagent configurations presently being used in medical, veterinary, food and agricultural immunodiagnostics all represent different ways of visualizing the primary antibodyantigen reaction. Over the past 25 years this methodology has been successfully applied to many of the analytical challenges of the medical health care industry for rapid and accurate measurement of analytes such as hormones, microorganisms, therapeutic drugs, drugs o f abuse, and tumor markers . The past 10 years has seen a rapid expansion of immunoassay techniques into forensic, veterinary, food and agricultural analyses. The potential of rapidly measuring a very minute quantity of a specific analyte from within a complex sample matrix, often with little or no sample clean-up, is one of the attractive features that has led to the widespread application of immunoassay techniques. Antibody-producing cells generally respond to macromolecules and compounds with a molecular weight greater than 10,000 Daltons when those materials are recognized as foreign by the immune system. In general, compounds of the molecular weight of most pesticides w i l l not independently elicit an immune response. If those compounds are covalently attached to a carrier macromolecule, however, the immune system w i l l respond and produce antibodies to the small molecule portion o f the complex (hapten) as well as to other regions of the carrier. The specificity o f an antibody to a small molecule such as a pesticide can be influenced to a large degree by the design of the immunogen used to induce antibody formation. The immunogen is constructed by covalently coupling the small molecule or a related analogue to a carrier protein. In general, antibody specificity is highest for the part o f the molecule furthest from the carrier protein. Thus i n synthesizing the immunogen it is possible to orient the small molecule in ways that w i l l favor antibody specificity to particular portions o f the molecule. Through selective construction of the immunogen, it is possible to induce antibodies that may or may not, for example, differentiate a parent pesticide from its major metabolite, or a specific pesticide from a related family of pesticides. Additional levels of specificity may also be achieved through selection o f appropriate monoclonal antibodies. Most small molecules, including many of the pesticides, in the less than 1000 Dalton molecular weight range only have one antigenic determinant and thus must be analyzed using a competitive immunoassay format. A wide variety o f competitive immunoassays have been developed for analysis of small molecules such as antibiotics (5), pesticides (6), toxins (7), and hormones (8). Further information concerning basic immunoassay technology is covered i n detail i n Chapter 1.
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IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS
Alachlor Immunoassay Development The herbicide alachlor is one of the most widely used pesticides i n North America. It is used primarily to control grassy weeds in corn and soybeans and can be found as a groundwater contaminant. Some laboratory testing services routinely analyze water samples for alachlor residues using chromatographic methods. The availablility of an appropriate immunoassay would reduce the turnaround time and costs incurred in current residue analyses for alachlor and enable larger numbers of samples to be analyzed. Development o f a laboratory immunoassay for alachlor analysis was first described by Wratten and Feng (9). This paper describes work at Agri-Diagnostics toward the development o f a standardized and stabilized multiwell immunoassay kit and development o f a simple, rapid, and quantitative on-site immunoassay kit for analysis o f alachlor i n groundwater.
Experimental Methods Protein conjugates o f alachlor were synthesized by two different methods for use as immunogens and antibody screening conjugates. Conjugates were prepared using a carbodiimide driven condensation reaction of the 2-(4-aminophenylthio)-2\6 diethyl-N-methoxymethylacetanilide and bovine serum albumin ( B S A ) as shown in Figure 1. Alachlor was also coupled directly to thiolated chicken albumin and B S A through an alkylation reaction. Acetyl homocysteine thiolactone was used to first thiolate the protein which was subsequently mixed with alachlor under alkaline conditons resulting i n alkylation of the thiol by the herbicide to form the conjugate (Figure 2). These conjugates were used to immunize sheep and mice and also to coat 96 well polystyrene plates for use in antibody screening. Alachlor was also covalently coupled to horseradish peroxidase for use as an enzyme-hapten tracer in a direct competitive assay format. Alachlor, metolachlor, and butachlor were obtained from Chem Service (West Chester, Pa). Acetanalide anlogues used i n the cross-reativity studies were provided by Ricerca, Inc. (Painesville, Ohio). Indirect and direct competitive multiwell assay formats, and a rapid field usable format were developed and performed as described below: f
Indirect competitive multiwell assay: 1. A d d 50|il o f negative control, standards, and test samples to wells o f multiwell plates coated with the chicken albumin-alachlor conjugate. 2. A d d 50\i\ of alachlor antibody to each well and mix for 10 minutes. 3. Rinse out wells five times and add 100|il of anti-sheep globulin peroxidase to each well. 4. Rinse out wells five times and add lOOjil o f enzyme substrate ( A B T S ) to each well. M i x for 10 minutes. 5. A d d 50^.1 stop solution to each well and m i x for 10 seconds. 6. Read absorbance at 405nm.
Direct competitive multiwell assay: 1. Make a 1:1 mixture of the water sample or standard or negative control with the peroxidase-alachlor tracer. 2. A d d lOOjil of each mixture from step 1 into respective wells o f multiwell plates coated with purified sheep anti-alachlor antibody and mix for 10 minutes.
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CH>CH
3
ALACHLOR AMINOBENZENE + BSA-COOH +
UREA DERIVATIVE
Figure 1.
Conjugation o f alachlor aminobenzene to bovine serum albiimin.
IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS
CH2CH3
CH^CHg
*
ALACHLOR + OA-NH
1
2
L- NHCOCH3
ACETYL HOMOCYSTEINE THIOLACTONE
J
—
HCL
CHICKEN ALBUMIN - ALACHLOR CONJUGATE
Figure 2. Conjugation of alachlor to chicken albumin using acetyl homocysteine thiolactone.
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Rinse out wells five times and add 100|il o f enzyme substrate ( A B T S ) to each well. M i x for 10 minutes. A d d 50|il stop solution (1.5% NaF) to each well and mix for 10 seconds. Read absorbance at 405nm.
Rapid field usable assay: 1. M a k e a 1:1 mixture of the water sample or standard or negative control with the peroxidase-alachlor tracer. 2. A d d 1 drop of the negative control peroxidase-alachlor tracer mixture and one drop of the sample peroxidase-alachlor tracer mixture to respective antibody coated zones on the surface of the porous plastic device. 3. A l l o w all liquid from each drop to completely drain into the porous device. 4. A d d 1 drop of rinse solution to each zone and allow to drain. 5. A d d 1 drop of enzyme substrate solution to each zone and allow to drain. 6. A d d 1 drop o f stop solution to each zone and allow to drain into device 7. Quantitate results using hand-held reflectometer. Total test time approximately 5-10 minutes Cross-reactivity is expressed as the ratio of the concentration of alachlor to each test compound at level that gives 50% inhibition of the immunoassay maximal binding level. R e s u l t and Discussion A n indirect competitive assay was developed using the chicken albumin-alachlor conjugate as the solid phase antigen and antisera obtained from a sheep immunized with the BSA-alachlor aminobenzene immunogen. A sensitivity limit of approximately 1 ppb was observed. Cross-reactivity with two other acetanalide herbicides, metolachlor and butachlor, was 2.3% and 6.4% respectively, indicating that the methoxymethyl region of the alachlor molecule plays a significant role i n antibody specificity (Figure 3 ). Additional sensitivity was obtained by formatting the assay in a direct competitive configuration where the antibody was immobilized to the solid phase and a peroxidase-alachlor conjugate was used in a simultaneous competition reaction with the sample. A sensitivity limit of approximately 0.1 ppb was observed with an IC50 of about 2ppb. The dynamic range of the assay allows for quantitation as high as 50ppb (Figure 4 ) . Cross-reactivity of the direct competitive assay with metolachlor and butachlor was 0.5% and 1.0% respectively. Cross-reactivity analysis of a variety of other chloroacetanalides indicated that changes to alachlor at either the methoxy methyl side chain or to the ethyl groups on the ring result in a major loss of antibody recognition (Table 1). Field Vsflfrle Format On-site immunoassay testing formats are regularly used in the medical area for doctors office and home testing applications. One type of on-site format that has become very popular for home pregnancy testing (10) and one which Agri-Diagnostics has successfully used for plant disease diagnostics (11) and is now applying to chemical analysis, is the flow through assay device. In this format the antibody or antigen is immobilized within a microporous surface that is in contact with an absorbant reservoir. The test sample is added to the surface o f the device and flows through the activated area into the absorbant reservoir. The movement of the sample and the subsequent reagents across the immobilized antibody or antigen
34
IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS
100
-10 H .1
1
'
I 1
•
•
M l |
•
10
I
I
II M l l |
100
•
1000
I
I I II l l |
10000
100000
[Acetanalide] ng/ml Figure 3. Comparative dose response curves of alachlor, butachlor, and metolachlor using an indirect competitive ELISA.
[Alachlor] ng/ml Figure 4. Alachlor dose response curve using a peroxidase-alachlor conjugate in a direct competitive assay configuration.
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Table 1 CROSS-REACTIVITY OF ACETANALIDE ANALOGUES IN THE DIRECT COMPETITIVE ALACHLOR IMMUNOASSAY
Compound Alachlor
I 50 Value (ug/ml) 0.002
Structure
% Cross-Reactivity 100.0%
CHCH CH.CH, O Butachlor
0.20
1.0%
CCHp
(oy< VrV
Metholachlor
0.40
0.5%
SDS-023018
0.05
4.0%
^CH.OCH^^CH,
CH^H,
"^CHCHOCH CH, CH /-T
