Immunoassay for pesticides and their degradation products

Details Immunoassay for pesticides and their degradation products Immunoassay for pesticides and their degradation products

2000-07-21

2003-10-21

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007940, C436S527000, C436S531000, C436S546000, C436S815000, C530S404000, C530S405000, C530S389500

Reexamination Certificate

active

06635434

ABSTRACT:

FIELD OF INVENTION
The present invention relates to a novel hapten-polymer carrier complex comprising a linking unit located so as to not disrupt the antigenic determinant groups of the hapten. The invention further relates to a specific immunoassay for the identification and quantification of pesticides and their degradation products using said hapten.
GENERAL BACKGROUND
Pesticides are widely used in many countries. Dichlorbenil (Casoron G®, dichlorobenzonitrile) is a pesticide used as a broad-spectrum herbicide both in agriculture and in urban areas. This herbicide is degraded, mainly to 2,6-dichlorobenzamide (BAM) by biotic or abiotic processes in the hydrosoil (
FIG. 1A
) (Beynon and Wright, 1972; Montgomery et al., 1972; Verloop, 1972). Chlorthiamid (Prefix®), a commercial pesticide, also degrades to BAM (FIG.
1
D). BAM is very persistent, and is now contaminating a large number of ground-water areas in Europe and the U.S.A.(Brüsch, 1998). To prevent further leaching of BAM to the drinking water, dichlorbenil is now banned in many countries.
Most BAM-analyses are based on high-performance liquid chromatography (HPLC) and gas chromatography (GC) (van Rossum et al., 1978; Connick and Bradow, 1984) which have detection limits of approximately 0.01 &mgr;g/L. Such analyses are very costly for national groundwater-monitoring programs. To lower expenses on pesticide analyses, immunoassays have been developed for several pesticides (Kaufman and Clower, 1995; Issert et al., 1997), but not yet for BAM or the degradation products of dichlorbenil.
The advantage of immunoassays is their extremely high sensitivity, which enables the quantification of pesticides or their degradation products in concentrations lower than 1 &mgr;g/L without the need for concentration of samples. Analyses based on HPLC and GC (and GC mass spectroscopy, GC-MS) often require concentration of the sample by evaporation of approximately 2 L of aqueous sample to volumes one thousandth of the original volume.
An immunoassay involves the detection or measurement of an analyte using the specific interaction between an antibody and an antigen. The immune complex that occurs specifically between the antigen and the antibody forms the foundation of such assays. Usually, the antibody is labelled with a tag for detection or quantification purposes. An assay comprising of an enzyme-linked antibody (or antigen) complexing to an immobolised antigen (or antibody) is commonly referred to as an ELISA (enzyme-linked immunosorbent assay).
An immunological process for the assaying a class of herbicides have previously been described (EP 0 300 381 B1). However, this class of compounds does not foresee the use of an appropriate linking unit moiety, nor does it anticipate the class of compounds or haptens described herein. It describes an immunological process for the assay of tri-substituted aromatic rings in which two of the three substituents are fixed 1,3 with respect to one another (meta to one another) whereas the third substituent can be in positions 2, 4, 5, or 6 (ortho or para to either of the other two substituents). Moreover, any of the three substituents can serve to couple to a solid matrix. The hapten, which is the same as the hapten immobilised to the matrix, was used to raise antibodies A, B, or C for the purpose of the assay and that at least two of the said antibodies were reacted separately with a sample supposedly containing said hapten.
Hammock et al (
J. Agric. Food Chem.
1984, 32, 1294) investigated the sensitivity and specificity of immunoassays for benzoylphenylureas in relation to heterologous site systems and homologous site systems whereby the coupling site of the hapten was varied or remained constant, respectively.
In U.S. Pat. No. 5,654,178, an antibody useful in an immunoassay for tetrachloroisophthalonitrile is raised. The hapten is coupled to protein via a functional group interchange of a chloro group present on the ring.
SUMMARY OF THE INVENTION
A first aspect of the invention relates to a hapten-polymer carrier complex comprising a polymer carrier and a compound of the general formula I
wherein R
2
serves as a linking unit to the polymeric carrier to form the hapten-polymer carrier complex; and wherein R
1
is selected from the group consisting of —C(═O)—N(R
5
)(R
5
, —N(R
5
)—C(═O)—OR
5
, —C(═O)—OR
5
, —N(R
5
)—C(═O)(R
5
, —N(R
5
)—C(═S)(R
5
, —C(═S)—n(R
5
)(R
5
, —N(R
5
)—C(═S)—N(R
5
)(R
5
, —N(R
5
)—C(═O)—N(R
5
)(R
5
, —O—C(═O)—R
5
, —C(═S)—OR
5
, —o—C(═S)—R
5
, —O—R
5
, —N(R
5
)(R
5
, —S—R
5
, —CH
2
—R
5
, —NC, and halogen; where R
5
is independently selected from the group consisting of hydrogen, optionally substituted C
1-4
alkyl, optionally substituted C
1-6
alkenyl, optionally substituted C
2-6
alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C
3
-C
7
cycloalkyl; and wherein X
1
and X
2
are independently selected from halogens.
In a second aspect, the invention relates to a use of said complex for the identification or quantification of a pesticide and/or its degradation product or products by an immunoassay involving antibodies raised from said hapten-polymer carrier complex.
A second aspect of the invention relates to a method, for the identification or quantification of a pesticide and/or its degradation product or products by an immunoassay involving antibodies raised from a hapten-polymer carrier complex of general formula I,
wherein X
1
,X
2
, R
1
, R
2
are as defined as above; and
a compound of the general formula II immobilised to a solid support
wherein Z
1
is C—R
6
, Z
2
is C—R
7
, Z
3
is C—R
8
and Z
4
is C—R
8
, where at least one of R
6
-R
10
is present and serves as a linking unit for immobilisation of the hapten to the solid support, and the ones of R
6
-R
10
which are present and which do not serve as a linking unit are independently selected from the group consisting of —C(═O)—NH
2
, —N(R
5
)(R
5
), —CN, N(R
5
)—C(═O)(R
5
), —N(R
5
)—C(═S)(R
5
), —C(═S)—N(R
5
)(R
5
, —N(R
5
)—C(═S)—N(R
5
)(R
5
), —N(R
5
)—C(═O)—N(R
5
)(R
5
), —N(R
5
)—C(═O)—OR
5
, —C(═O)N(R
5
)(R
5
), —C(═O)—OR
5
, —O—C(═O)—R
5
, —C(═S)—OR
5
, —O—C(═S)—R
5
, hydrogen, and halogen; and wherein those of R
6
-R
10
which serve as a linking unit are selected from the group consisting of —C(═O)—N(R
5
)—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, —N(R
5
)—C(═O)—CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, —O—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, —N(R
5
)—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, —S—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, —(CH
2
)
n+1
—R
3
—(CH
2
)
m
—R
4
, —C(═O)—O—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
, and —O—C(═O)—(CH
2
)
n
—R
3
—(CH
2
)
m
—R
4
; wherein n and m are independent and are integers from 0 to 8; R
3
is selected from the group consisting of —O—C(═O)—, —C(═O)—O—, —O—C(═S)—, —O(—S)—O—, —N(R
5
)—C(═O)—, —C(═O)—N(R
5
)—, —O—, —S—, —N(R
5
)—, optionally substituted C
1-4
alkylene, optionally substituted arylene, optionally substituted heteroarylene, an optionally substituted C
3
-C
7
cycloalkylene, R
4
is selected from the group consisting of activated forms of a carboxyl, carboxylic acid, amine, acyl halides, activated esters, thiols, disulphides (—S—S—R), halides, sulphonates, quinones and imides; and R
5
is independently selected from the group consisting of hydrogen, optionally substituted C
1-4
alkyl, optionally substituted C
2-5
alkenyl, optionally substituted C
2-6
alkynyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C
3
-C
7
cycloalkyl.
A third aspect of the invention relates to kit for an immunoassay comprising a solid support and hapten-polymeric carrier complex as defined above immobilised to said solid support.
DETAILED DESCRIPTION OF THE INVENTION
The present investigators have surprisingly found that in immunoassays for certain 1,3-dihalo aromatic pesticides, such as dichlorbenil, chlorthiamid and their derivatives and degradation pro

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