Chapter 16
Competitive- and Inhibition-Type Immunoassay for Determination of Endosulfan
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Bernhard Reck and Jürgen Frevert Batelle-Institut e.V., Am Römerhof 35, 6000 Frankfurt am Main 90, Federal Republic of Germany
Two different types of immunoassays, a competitive-type and an inhibition-type, were developed and compared for the insecticide, endosulfan. The detection range of both assay types was similar, 3-500 ng/ml for the competitive-type and 5-500 n g / m l for the inhibition-type assay. Metabolites of endosulfan and other insecticidal chlorohydrocarbons possessing a hexachlorocyclopentene structure exhibited considerable crossreaction in both assays, so these immunoassays could be used for detection of class-specific compounds. The inhibition-type immunoassay showed less susceptibility to interfering factors and, therefore, seemed to be more suitable for environmental analysis. Conventional analyses for the insecticide, endosulfan, in environmental samples requires time-consuming multistep cleanup procedures prior to the final quantification by gas chromatography (1). For this reason, a sensitive and rapid ELISA could be of great value if large numbers of samples have to be analyzed. The advantages of immunoassays for residue analysis have been described by Hammock and Mumma (2). To develop such an immunological assay for endosulfan, specific antibodies directed against endosulfan are necessary. As a low molecular weight substance, endosulfan per se is not immunogenic, i.e. it is not capable of inducing the synthesis of antibodies in animals. The first step in the ELISA development is, therefore, the covalent coupling of endosulfan or a derivative to a carrier protein. Therefore, a derivative of endosulfan, endosulfandiol, was linked to a high molecular weight protein, Keyhole limpet hemocyanin (KLH) by first synthesizing the hemisuccinate and then the N-hydroxysuccinimid 0097-6156/90ΑΜ42-0193$06.00Λ) © 1990 American Chemical Society Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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ester. This active ester readily reacts with free amino groups of the K L H (3), producing an immunogen which was then used to raise specific antibodies in rabbits. For the competitive-type ELISA (description, see below), endosulfan or a derivative must also be conjugated to an enzyme. To avoid the antibodies from reacting with the linkage between endosulfandiol and K L H , another coupling procedure was chosen. Endosulfandiol was linked to ethylenediamine utilizing carbodiimidazole such that a free amino group was available. The vicinal hydroxy groups of the glycoprotein enzyme, horseraddish peroxidase, were oxidized with N a I 0 4 , resulting in the formation of aldehyde groups (4). These aldehyde groups then reacted with the free amino group of the endosulfandiol coupled to the ethylenediamine to produce the enzyme conjugate. Because of the strongly hydrophobic character of endosulfandiol, the synthesis of the enzyme conjugate was the most critical step in the development of the immunoassay. When horseraddish peroxidase was linked to several endosulfan derivatives per molecule, it became very insoluble and lost its enzymatic activity. A n enzyme conjugate with a ratio of 1:1 was most favorable for the immunoassay. On the other hand, a KLH-endosulfan conjugate with a high number of endosulfan per K L H was insoluble and was an effective immunogen producing a high antibody titer. Using these reagents, the inhibition and the competitive type immunoassay were established and their characteristics are compared in Table I. Table I. Comparison of Competitive- and Inhibition-Type Immunoassay
incubation steps time required detection limit detection range consumption of antisera stability of reagents susceptibility to interfering factors in environmental samples
Competitive-type immunoassay
Inhibition-type immunoassay
1 1.5 hr 3 ng/ml 3-500 ng/ml high
2 >5 hr 5 ng/ml 5-500 ng/ml low
peroxidase-endosulfan very unstable high (inhibition of enzyme activity)
no unstable reagent low (inhibition of antibody-antigen binding)
Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
16. RECK AND FREVERT
Compétitive- and Inhibition- Type Immunoassay 195
In the competitive-type immunoassay, the antibody was immobilized in the wells of a polystyrene microtiter plate (5). The sample to be analyzed, which potentially contained endosulfan, was then incubated with a fixed amount of peroxidase-endosulfandiol conjugate. Free endosulfan and the endosulfandiol enzyme conjugate competed for binding to the immobilized antibody. The wells were then washed and the chromogen, o-phenyldiamine, was added. The optical density of the color of the solution in the wells was determined. The intensity of the color was proportional to the amount of bound peroxidase conjugate. A standard curve derived from the analysis of a serial dilution of endosulfan was used for the basis of analysis of samples. Figure 1 gives an example of a standard curve and demonstrates how variation of the enzyme-conjugate influences the measuring range of the assay. For comparison of different test runs, the A / A ratio was plotted versus endosulfan concentration (Amax s the optical density of the test solution without the analyte). The detection limit of the assay was lowered by reducing the concentration of the peroxidase conjugate. On the other hand, lowering the concentration of the enzyme conjugate reduced the absorbance which resulted in a decreased sensitivity. Therefore, one had to identify the optimal concentration for the enzyme conjugate. A peroxidase conjugate concentration of 150 ng/ml was used to achieve a sufficiently high absorbance. The final competitive-type ELISA resulted in measuring endosulfan concentrations in the range of 3-500 ng/ml. m a x
w a
A major drawback of competitive-type immunoassay arose when environmental samples were analyzed. In this procedure, the enzyme conjugate was added directly to the sample, thus, all substances in the sample which have an effect on the enzyme would interfere with the assay. This dramatically limits the use of the competitive-type ELISA assay. Therefore, an inhibition-type ELISA was developed which would supposedly be less susceptible to interfering factors present in the sample. In this type of assay, a protein-endosulfandiol conjugate is immobilized in a microtiter plate. In the first step, a limited, constant amount of antibody was incubated with the endosulfan containing sample. This can be done in the microtiter plate or during an external incubation step, i.e., sample and antibody were preincubated in a tube. Antibodies which had not reacted with endosulfan would bind to the immobilized endosulfandiol conjugate on the test plate. The amount of bound antibody, which was dependent on the amount of endosulfan present in the sample, was then quantitated with a second, enzyme-labeled antibody directed against the first antibody. Because of the washing steps between the incubations, the enzyme is not effected by potentially interfering substances present in the sample.
Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL ANALYSIS
Standard curves for different incubation procedures are shown in Figure 2. Only minor differences in sensitivity were found and this allows a variable test setup in respect to preincubation. No appreciable differences were demonstrated between the competitive- and inhibition-type immunoassays (Fig 2). In contrast, differences in the crossreaction with endosulfan derivatives relative to the two type assays were found. Because the coupling of an endosulfan derivative was achieved by using a hydroxyl group of endosulfandiol, the hexachloropentene structure was exposed, and a strong crossreactivity with all compounds having this structure could be anticipated. Table II shows that all derivatives of endosulfan (except endosulfanlactone in the competitive-type immunoassay) exhibited a significant affinity to the antiserum. However, there were major differences in the amount of crossreaction in the assay types, e.g., endosulfandiol was better recognized in the competitive-type immunoassay than in the inhibition-type assay. A possible reason for these differences may be that in one case the antibodies were immo bilized and the binding of these antibodies to the solid phase could have an effect on the avidity.
Table Π. Crossreaction of Endosulfan Derivatives Pesticide or Derivative ct-Endosulfan β-Endosulfan Endosulfandiol Endosulfanether Endosulfansulfate Endosulfanlactone
% Crossreaction Inhibition-type Competitive-type 100 128 34 140 131 11
100 100 158 57 203 0.02
The behavior of other insecticidal chlorohydrocarbons has also been investigated, and the crossreactions of these are presented in Table III. A l l compounds possessing a hexachlorocyclopentene structure showed a significant reactivity in both assay types. Surprisingly, both immunoassay had a higher sensitivity for endrin than for endosulfan. The stereochemical analogue of endrin, dieldrin, however, showed only moderate affinity to the antibody. Because of this crossreaction, neither type of assays, developed with this antiserum, allows for specific detection of endosulfan itself, but can be used for screening for the presence of a class of chlorohydrocarbons.
Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
16. RECK AND FREVERT
120
Competitive- and Inhibition-Type Immunoassay 197
%A/A max
100
10
100
1000
10000
Endosulfan C o n c e n t r a t i o n in ng/ml C o n j u g a t e 625ng/ml
Conjugate 3 0 0 n g / m l
C o n j u g a t e 150ng/ml
Conjugate
Figure 1.
120
75ng/ml
Standard curves for endosulfan at varying concentrations of endosulfandiol-horseradish peroxidase conjugate.
%A/A max
100f
10
100
1000
10000
Standard C o n c e n t r a t i o n in ng/ml O V E R N I G H T , 279 Κ
Figure 2.
1 H O U R , 310 Κ
NO
PREINCUBATION
Standard curves for endosulfan at varying times of the antibody-sample preincubation step.
Van Emon and Mumma; Immunochemical Methods for Environmental Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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IMMUNOCHEMICAL METHODS FOR ENVIRONMENTAL
ANALYSIS
Table ΙΠ. Crossreaction of Insecticidal Chlorohydrocarbons Correlated to a-Endosulfan (100%) Pesticide
Aldrin Alodan Heptachlor Heptachloroepoxide Endrin Dieldrin Lindane Kelevan
a
% Crossreaction Inhibition-type Competitive-type 14 6 12 20 364 41 2
