WO2000014538A1 - Linker-assisted immunoassay for glyphosate - Google Patents

Abstract

This invention provides a simple, inexpensive and high throughput assay method for the widely used herbicide, glyphosate, as an alternative to the currently used complex analytical methods. The invention provides a number of immunological reagents suitable for use in an immunoassay for glyphosate. The reagents include: (i) immunogens prepared by covalently linking glyphosate or glyphosine to a suitable carrier protein such as porcine thyroglobulin (TG) via the carbodiimide reaction, (ii) a phase coating conjugate prepared by covalently linking glyphosate to a suitable carrier protein such as BSA and (iii) antibodies to the above-mentioned immunogens. The invention also provides an immunoassay method with enhanced sensitivity (ppb level) for the analysis of glyphosate in environmental or biological samples. Methods for protein-hapten conjugate preparation, anti-glyphosate antibody production, and pre-assay derivatization of samples applicable to a variety of immunoassay formats are also provided.

Description

I NKER-ASSISTED IMMUNOASSAY FOR GLYPHOSATE

FIELD OF THE INVENTION

The present invention generally relates to assays for determining levels of compound(s) in a liquid medium. More particularly this invention relates to methods for determining the level of glyphosate in water at concentrations as low as the parts per billion (ppb) level.

ACKGRQUND QF THE INVENTION

The global use of glyphosate-containing herbicides creates a continuous need for the measurement of glyphosate in a variety of matrices. Current methods for glyphosate analysis are tedious and require sophisticated equipment.

Glyphosate was introduced by Monsanto Company as a new herbicide in 1974 and is now sold globally under numerous trademarks, including Roundup™ herbicide. Chemically, glyphosate (N-phosphonomethylglycine; C₃H₈NOsP; M.W., 169.1) is a small zwitterionic amino acid derivative that poses unique problems in the development of analytical methods. Due to the small size of the analyte and its insolubility in common organic solvents, most analytical methods for glyphosate involve extensive sample cleanup, derivatization, and separation on gas or liquid chromatography columns. These chromatographic techniques are slow and cumbersome and require sophisticated equipment. An excellent overview of current analytical methods for detecting glyphosate can be found in Franz, et al, Glyphosate: A Unique Global Herbicide, American Chemical Society Monograph 189, pp 80-97, Washington, DC, 1997.

In 1974, Congress passed the Safe Drinking Water Act. This law requires the Environmental Protection Agency of the United States Government, the EPA, to determine safe levels in drinking water of chemicals that do, or may, cause health problems. These levels are called Maximum Contaminant Level Goals (MCLG). the MCLG for glyphosate has been set at 7 ppb because the EPA believes this level would not cause any of the known or suspected potential health problems. In addition, the EPA believes, given present technology and resources, this is the lowest level to which water systems can reasonably be required to remove this contaminant from drinking water supplies.

Therefore, the development of a simple, rapid, cost-efficient immunoassay method for glyphosate is highly desirable. However, a major challenge to developing a successful immunoassay for a small analyte, such as glyphosate, is the difficulty of generating analyte-specific antibodies having sufficiently high affinity toward the analyte to be useful in such assay protocols as a competition enzyme-linked immunosorbent assay (ELISA).

Since small molecules are "overlooked" by the immune system, it is common to enhance the immunogenicity of low molecular weight analytes (haptens) (100-200 Daltons) by covalently conjugating the analyte, or its analogue, to an immunogenic carrier protein. However, it is now widely realized that antibodies raised against such conjugates rarely have sufficient affinity for the unconjugated hapten, which is the analyte of interest, to be useful for most immunoanalytical purposes. It is believed that the surface area of the analyte is insufficient for optimal interaction with the antibody combining site of the antibody molecule (Chappey et al, Journal of Immunological Methods 172:219-225. 1994). Generally, this phenomenon is due to the antibody having been originally selected to recognize the hapten as well as the linkage arm binding the hapten to the carrier protein in the immunizing conjugate. Thus, the design for the synthesis of protein-hapten conjugates has a profound influence on the specificity and sensitivity of an assay in which the antibody is used.

One method for overcoming this problem is to enhance affinity for a small analyte by chemically increasing the size of a hapten in the sample to be tested through formation of chemical derivatives. U.S. Patent No 4,818,683 to Morel and Delaage (1989) discloses an immunoassay method for monoamines based on chemical conversion in a sample intended for analysis, of a monoamine analyte, such as Wstamine, into a chemical derivative of higher molecular weight. Succinyl glycinamide (SGA) derivatives of the monoamine analyte, formed by acylation, incorporate the SGA moiety into the analyte in a test sample prior to assay of the sample to detect the presence of the analyte.

However, this prior art method is synthetically complicated since it requires reaction of samples with a novel acylation reagent, such as N-hydroxysuccinimide- ester-succinyl-glycinamide, to form a chemical derivative of the monoamine analyte in the sample prior to testing. Further, the success of this method requires a procedure that is slightly more complicated than the standard procedure for synthesis of the immunizing conjugate used to raise antibodies to be employed in the immunoassay. The immunizing conjugate is also a succinyl glycinamide (SGA) derivative formed by acylation of the monoamine analyte and purification of the derivatized analyte is required prior to conjugation of the derivative to a carrier protein to increase the immunogenicity of the conjugate.

For nearly four decades, immunoassay procedures have provided sensitive diagnostic tools for the in vitro detection of a variety of antigens, including those associated with disease or other physical conditions of clinical significance. These procedures are now being used at an accelerated pace for the detection and quantitation of pesticides in various biological and environmental samples. There are many variations on the ways in which immunoassays can be performed. Three classes of immunoassays are commonly used, the antibody capture assay, the antigen capture assay, and the two-antibody sandwich assay. In an antibody capture assay, the antigen is attached to a solid support, and labeled antibody is allowed to bind. After washing, the assay is quantitated by measuring the amount of antibody retained on the solid support. In an antigen capture assay, the antibody is attached to a solid support, and the labeled antigen is allowed to bind. The unbound components are removed by washing, and the assay is quantitated by measuring the amount of antigen that is bound. In a two-antibody sandwich assay, one antibody is bound to a solid support, and the antigen is allowed to bind to this first antibody. The assay is quantitated by measuring the amount of a labeled second antibody that can bind to the antigen. Generally the sandwich assay is not applicable to a small analyte such as glyphosate because of its inability to serve as a binding partner for both of the antibodies simultaneously. The general procedures and rationale for selecting one type of assay over another are well known in the art and are summarized in Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1988, Chapter 14, which is incorporated herein by reference).

Heterogeneous assays use a polyclonal antibody preparation bound to the solid phase. In these assays, a solution of labeled antigen is allowed to compete directly for the solid phase antibody with antigen in the sample being analyzed. Alternatively, a solution of labeled antigen can be added to the antibody in a sequential process. The extent to which the labeled antigen is bound to the solid phase, or is detected in the liquid phase, can be used as a measure of the presence and quantity of antigen in the sample being analyzed. Immunoassay procedures modified to use monoclonal antibodies are also known in the art. For example, U.S. Pat. No. 4,376,110 describes two-site immunometric assays using pairs of monoclonal antibodies, one bound to a solid phase and the other labeled to permit detection. The use of monoclonal antibody pairs which recognize different epitopic sites on an antigen has made it possible to conduct simultaneous immunometric assays in which the antigen and labeled antibody incubations do not require the intermediate washing steps of prior processes.

In view of the above state of the art and interest in developing assays for detection of concentrations of low molecular weight molecules, there is a need in the art for new and better methods for routine analysis of glyphosate utilizing a simple, high throughput immunoassay format.

BRIEF DESCRIPTION OF THE INVENTION

The present invention overcomes many of the problems in the art by providing methods for simple, inexpensive and high throughput assay of the widely used herbicide, glyphosate. One object of the present invention is to provide analyte-specific antibodies against glyphosate and chemically similar compounds for use in immunoassays, such as ELIS A, through a simple approach to immunogen preparation that does not require derivatization of the hapten prior to its conjugation to an immunogenic protein. A further object of the present invention is to provide a highly specific and sensitive (ng/ml level) linker-assisted immunoassay method for glyphosate in test samples, such as drinking water, extracts of soils, and the like.

These objects of the invention are met by providing (i) strategically designed protein-hapten conjugates for use as immunogens and as solid-phase-coating antigens in ELISA-based procedures, and (ii) a simple affinity-enhancing analyte derivatization procedure that enhances assay sensitivity, for example, providing an increase in assay sensitivity of up to 10⁴-fold or greater. Invention immunoassay method(s) require a simple pre-assay derivatization step during which the analyte is covalently attached to a linker moiety, such as glutaric acid or succinic acid, and use commercially available, inexpensive, and relatively stable reagents.

Therefore, in one embodiment according to the present invention, there are provided methods for obtaining an anti-glyphosate antibody. Invention antibody production method(s) comprise preparing an immunogenic conjugate by covalently coupling glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, directly to a first carrier molecule, immunizing a susceptible host at variable intervals with the conjugate; and obtaining the antibody from the host. When the hapten is a derivative containing at least two ionizable acidic groups, or salt(s) thereof, the coupling step is conducted under conditions selected to preserve the chemical identity of the at least two ionizable acidic groups in the derivative.

In another embodiment according to the present invention, there are provided linker-assisted immunoassay methods for the detection of glyphosate, or a salt thereof, in a test sample. Invention linker-assisted immunoassay method(s) comprise reacting the test sample with a linker having an activated carboxylic group to obtain an analyte-linker conjugate, contacting the reacted test sample with at least one invention anti-glyphosate antibody, further contacting the test sample containing the analyte-linker conjugate with a solid phase having immobilized thereon a solid phase coating conjugate comprising a second carrier molecule covalently coupled to glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, removing unbound components from the solid phase, and detecting the presence of bound anti-glyphosate antibody. The amount of bound antibody is inversely related to the amount of glyphosate, or a salt thereof, in the test sample. The carrier molecule in the coating conjugate, however, is not identical to (i.e., different than) the carrier molecule in the immunogenic conjugate used to obtain the anti-glyphosate antibody. When the hapten is a glyphosate derivative having at least two ionizable acidic groups in the derivative, the coupling step is performed under conditions selected to preserve the chemical identity of the at least two ionizable acidic groups in the derivative.

In another embodiment according to the present invention, there are provided test kit(s) for the immunochemical detection of glyphosate, or a salt thereof, in a test sample. Invention test kit(s) comprise a solid phase and at least one invention anti-glyphosate antibody, which is bound, or can be bound, to the solid phase. The test kit may further optionally comprise such additional reagents as a labeled hapten conjugate that binds to the antibody to create a labeled anti-glyphosate antibody, and a linker such as, for example, aspartic, glutamic, succinic, glutaric, adipic, N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic, or poly-glutamic acids.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing the results of comparative ELISA tests for deteπriining the glyphosate concentration (ng/ml) in an aqueous sample by the invention linker-assisted immunoassay method(s) (-•-) and by standard ELISA technology (-A-) . The tests were conducted in parallel using identical plates and identical solutions ∞ntaining antibodies raised against a glyphosine-porcine thyroglobulin (TG) conjugate. The results are shown as relative absorbance at 450 nm. Relative absorbance was obtained by dividing the mean optical density value for each standard point by the mean optical density value of a standard containing no analyte.

Figure 2 is a graph showing the results of parallel comparative inhibition ELISA tests for determining the glyphosate concentration (ng/ml) in an aqueous sample using anti-TG-glyphosine antibody (-•-) or anti-TG-glyphosate (- -) antibody in linker-assisted ELISA tests wherein the analyte is derivatized by reacting it with glutaric anhydride. The results are shown as relative absorbance at 450 nm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that the sensitivity of an immunoassay, such as a competition ELISA wherein the analyte is a low molecular weight molecule, can be enhanced if the analyte in the sample is conjugated prior to assay with a linker molecule such that the analyte conjugate in the sample mimics the immunoresponsive portion of the hapten-protein conjugate used to raise antibodies prepared for use in the assay.

The glyphosate molecule contains three functional moieties (i.e., carboxylate, secondary arnine, and phosphonate) separated by two methylene groups. Under physiological conditions, the phosphonate and carboxylate of glyphosate are negatively charged, and hence would serve as immuno-dominant groups for this hapten. The conjugation procedures used in carrying out this invention are designed to ensure that the chemical functionality of both of these groups is preserved during the coupling of this hapten to a carrier protein, when an immunostimulatory conjugate is prepared to raise antibodies to be used in an immunoassay and when a coating conjugate or a signal-generating conjugate (for example, horseradish peroxidase- glyphosate) is prepared for use in the immunoassay.

In invention immunoassay method(s), analyte in the test sample is derivatized so as to make the derivative more closely mimic the epitopic site to which antibodies raised against the immunogenic conjugate will bind with enhanced affinity. In derivatizing analyte in the test sample prior to testing, care should be taken to assure that the ionic character of negatively charged groups in the analyte are preserved in the derivative.

Therefore, in one embodiment of the present invention, there are provided method(s) for obtaining an anti-glyphosate antibody. Invention antibody-producing method(s) comprise preparing an immunogenic conjugate by covalently coupling glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, directly to a carrier molecule, immunizing a susceptible host at variable intervals with the conjugate; and obtaining the anti-glyphosate antibody from the host. Derivatives of glyphosate that contain at least two ionizable acidic groups, or salts thereof, can substitute for glyphosate in invention immunogenic conjugate(s). The coupling step in invention anti-glyphosate producing method(s) is performed under conditions selected to preserve the chemical identity of the at least two ionizable acidic groups in glyphosate or its derivative.

More particularly, when the immunogenic conjugate is obtained by attaching a carrier molecule to glyphosate, or a salt thereof, the carrier molecule preferably has free carboxyl groups, and imide bonds are formed between the carboxyl groups of the carrier molecule and the secondary amino group of glyphosate to form an immunogenic conjugate having two negatively charged groups that mimic the negative charges of free glyphosate. In these circumstances, the coupling is generally achieved in a two-step process, by first activating the carboxyl groups on the carrier protein using an activating agent, such as l-e yl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC), followed by a nucleophilic reaction with glyphosate, or a salt thereof. Generally the glyphosate or glyphosate derivative contributes a secondary amino or carboxylic group, and the carrier molecule contributes a carboxylic or primary amino group toward formation of the linkage, (e.g., via an active ester or a water soluble carbodiimide). The preferred glyphosate salt is a sodium salt.

For example, the carboxyl groups on the carrier molecule can be activated with EDC for about 2-5 minutes at pH of about 5, followed by a nucleophilic reaction with glyphosate at alkaline pH in the presence of a molar excess (over EDC) of phosphate. The excess phosphate in this reaction serves to quench EDC, thereby preventing it from activating the carboxylic group of glyphosate. The acidic pH is preferably maintained between about 4 and about 6, and the alkaline pH is preferably maintained between about 7.5 and 9.5.

An alternative strategy for preparation of an immunogenic conjugate that will yield anti-glyphosate antibodies comprises the use of a glyphosate derivative having two ionizable acidic groups, as a surrogate for glyphosate. When a glyphosate derivative having at least two ionizable acidic groups, or a salt thereof, is used to obtain invention anti-glyphosate antibodies, coupling is preferably achieved in a two- step process, by first performing carbodiimide-mediated activation of the derivative under acidic pH conditions, and then reacting the activated carboxylic group of the derivative with the amino groups of the carrier molecule under alkaline pH conditions. The acidic pH is preferably maintained between about 4 and about 6, and the alkaline pH is preferably maintained between about 7.5 and 9.5.

For example, glyphosine (N,N-bis(phosphonomethyl)-glycine) is a glyphosate derivative containing one carboxymethyl and two phosphonomethyl groups attached to a tertiary a ino group. In the invention method, EDC-mediated coupling of the carboxyl group of glyphosine to the lysine groups of a carrier protein, such as porcine thyroglobulin (TG), leaves two negatively charged phosphonic acid groups to mimic the negative charges of free glyphosate. Additional non-limiting examples of suitable glyphosate derivatives having at least two ionizable acidic groups that can be used as the hapten in preparation of invention immunogenic conjugate(s) include N-pho-φhonomemyliminodiacetic acid, iminodiacetic acid, N,N- bis hosphonomethyl) amine, and the like.

Non-limiting examples of carrier molecules useful in preparation of invention immunogenic conjugate(s) include porcine thyroglobulin, bovine serum albumin, human serum albumin, ovdbumin, keyhole limpet hemocyanin, and the like. The presently preferred carrier molecules are proteins, such as porcine thyroglobulin (TG) or bovine serum albumin (BSA). The preferred molecular weight range for the carrier molecule is from about 100,000 to about 10,000,000.

Antibodies used in invention assay(s) can be polyclonal, monoclonal, or a functionally active fragment thereof. Mono- or poly-clonal antibodies to glyphosate, its salts, and glyphosate derivatives, are raised in appropriate host animals by immunization with invention immunogenic conjugate(s) using conventional techniques as are known in the art. The preparation of monoclonal antibodies is disclosed, for example, by Kohler and Milstein, Nature 256:495-7, 1975; and Harlow et al., in: Antibodies: a Laboratory Manual, page 726 (Cold Spring Harbor Pub., 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice, or other small mammals, such as rabbits, with a composition comprising an invention immunogenic conjugate whose preparation is disclosed above, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Barnes et al., Purification of Immunoglobulin G (IgG), in: Methods in Mol .Biol, Jϋi 79-104,1992). Antibodies of the present invention may also be derived from subhuman primate antibodies. General techniques for raising antibodies in baboons can be found, for example, in Golderiberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J. Cancer, 4^:310-314, 1990.

It is also possible to use anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')₂, and Fv that are capable of binding glyphosate, or a salt thereof, especially after the glyphosate or salt thereof has been derivatized with a linker molecule as disclosed herein. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')₂. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R.R., Biochem. J., 22: 119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and V chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat 7 Acad. Sci. USA 6_9_:2659- 62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and V_L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al, Science 242:423-426, 1988; Pack et al, Bio/Technology 11:1271-77, 1993; and Ladner et al, U.S. Patent No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2- 106-10, 1991.

In another embodiment according to the present invention, there are provided methods for obtaining strategically designed protein-hapten conjugates for use as solid-phase coating conjugates in invention ELISA-based assay(s) for detection of glyphosate, or a salt thereof. Preparation of a solid phase coating conjugate for use in invention assays comprises covalently linking glyphosate, a derivative containing at least two ionizable acidic groups, or salt(s) thereof, to a carrier protein that is different than (i.e., not identical to) the carrier protein used in obtaining antibod(ies) for use in invention ELISA-based assay(s). For example, if the carrier protein used in invention immunogenic conjugate(s) to obtain anti-glyphosate antibod(ies) is thyroglobulin, the carrier protein used in the coating conjugate is not thyroglobulin, but is selected from, for example, bovine serum albumin or ovalbumin. However, the coating conjugate mimics the ionic characteristics of the immunogenic conjugate to the extent that negatively charged groups in the glyphosate, a derivative containing at least two ionizable acidic groups, or salt(s) thereof, are preserved in the coating conjugate. In the practice of invention assay method(s), it is not necessary that the glyphosate derivative used in the coating conjugate be identical to a glyphosate derivative used to obtain invention immunogenic conjugate's).

In general, glyphosate derivatives suitable for use in the preparation of invention coating conjugate(s) can be chosen from N-phosphonomethylglycine (with the preferred linkage site being the secondary amino group thereof), N, N- bis(phosphonomethyl) glycine (with the preferred linkage site being the carboxyl group thereof), N, N-bis(phosphonomethyl) amine (with the preferred linkage site being the secondary amino group thereof), N-phosphonomethyl-iminodiacetic acid (with the preferred linkage site being at one of the two carboxyl groups thereof), iminodiacetic acid (with the preferred linkage site being the secondary amino group thereof), and the like. Preferred coating conjugates comprise N-phosphonomethylglycine covalently coupled to bovine serum albumin or N, N-bis(phosphonomethyl)glycine covalently coupled to ovalbumin.

Invention coating conjugate(s) and antibody(ies) are successfully employed in accordance with invention linker-assisted ELISA method(s) for the detection of glyphosate, or salt(s) thereof, in a test sample. Invention assay method(s) employ a novel derivatization step wherein the test sample is reacted with a linker having an activated carboxylic group to conjugate the linker with glyphosate, or salt(s) thereof, in the test sample. Attachment of the linker to the glyphosate in the test sample enhances the affinity of invention anti-glyphosate antibod(ies) for the glyphosate therein.

Therefore, in another embodiment according to the present invention, there are provided linker-assisted immunoassay method(s) for the detection of glyphosate, derivatives thereof containing at least two ionizable acidic groups, and salt(s) thereof, in a test sample. Invention linker-assisted immunoassay method(s) comprise:

(a) reacting the test sample with a linker having an activated carboxylic group to obtain an analyte-linker conjugate in the test sample,

(b) contacting the reacted test sample with at least one invention anti-glyphosate antibody,

(c) contacting the test sample containing the analyte-linker conjugate with a solid phase having immobilized thereon a solid phase coating conjugate comprising a second carrier molecule covalently coupled to glyphosate, a derivative containing at least two ionizable acidic groups, or salt(s) thereof,

(d) removing unbound components from the solid phase, and

(e) detecting the presence of bound anti-glyphosate antibody, wherein the amount of bound antibody is inversely related to the amount of the glyphosate, derivative thereof containing at least two ionizable acidic groups, or salt(s) thereof, in the test sample, and wherein the second carrier molecule in the coating conjugate is not identical to the carrier molecule in the immunogenic conjugate used to obtain the anti-glyphosate antibody.

Preferably, the anti-glyphosate antibody is contacted with the test sample at a pH of about 7 to about 10, and the analyte-linker conjugate is formed at apH of about 7 to about 10 so that an activated carboxylic group on the linker becomes attached to glyphosate in the test sample via the secondary amine group thereof. Suitable linkers for reaction with the test sample include succinic, glutaric, adipic, N-acetyl-aspartic, N-acetyl glutamic, poly-aspartic, and poly-glutamic acids, succinic and glutaric anhydrides, and the like. Generally, the linker is covalently linked to the secondary amino group of glyphosate, thereby enhancing the affinity of the first antibody for the glyphosate.

Invention linker-assisted assay method(s) may further comprise attaching a detectable label to the anti-glyphosate antibody on the solid phase. For example, the anti-glyphosate antibody can be conjugated to biotin and the detecting will comprise contacting the anti-glyphosate antibody with an enzyme-labeled molecule that binds strongly to the biotin. Alternatively, invention immunoassay method(s) can further comprise binding to the anti-glyphosate antibody on the solid phase a second antibody conjugated to a signal-generating agent, such as an enzyme, radioisotope, chemiluminescent or fluorescent label, colored microbead, colloidal gold, and the like.

Radioisotopes suitable for use as a signal-generating agent in the practice of invention immunoassay method(s) include tritium, carbon 14, phosphorous 32, iodine 125, iodine 131, and the like, which can be attached to an antibody by methods well known in the art. For example, ¹²⁵I can be attached to an antibody by procedures such as the chloramine-T procedure, or the lactoperoxidase procedure. These techniques plus others are discussed in H. Uan Vunakis and J. J. Langone, Editors, Methods in Enzymolgy, Vol. 70, Part A, 1980, which is hereby incorporated by reference. Chromogenic labels suitable for use as a signal-generating agent in the practice of invention immunoassay method(s) include compounds that absorb light in the visible or ultraviolet wavelengths, and the like. Such compounds are usually dyestuffs and include quinoline dyes, triarylmethane dyes, phthaleins, insect dyes, azo dyes, anthraquinoid dyes, cyanine dyes, phenazoxonium dyes, and the like.

Fluorogenic compounds suitable for use as a signal-generating agent in the practice of invention immunoassay method(s) include those that emit light in the ultraviolet or visible wavelength subsequent to irradiation by light, and the like. The fluorogens can be employed by themselves or with quencher molecules. The primary fluorogens are those of the rhodamine, fluorescein, and umbelliferone families. The methods of conjugation and use of these and other fluorogens can be found in the art. See, for example, J. J. Langone, H. Van Vunakis et al., Methods in Enzymology, Vol. 74, Part C, 1981, especially at page 3 through 105. For a representative listing of other suitable fluorogens, see Tom et al., U.S. Pat. No. 4,366,241, issued Dec. 28, 1982, especially at column 28 and 29. For further examples, see also U.S. Pat. No. 3,996,345, herein incorporated by reference.

Those skilled in the art will recognize that an enzyme-catalyzed signal system is, in general, more sensitive than a non-enzymatic system. Thus, for use in the practice of the present invention, catalytic labels are the more sensitive non- radioactive labels.

Catalytic labels are well known in the art and include single and dual ("channeled") enzymes such as alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and the like. Examples of dual ("channeled") catalytic systems include alkaline phosphatase and glucose oxidase using glucose-6- phosphate as the initial substrate. A second example of such a dual catalytic system is illustrated by the oxidation of glucose to hydrogen peroxide by glucose oxidase, which hydrogen peroxide would react with a leuco dye to produce a signal generator. A further discussion of catalytic systems can be found in Tom et al., U.S. Pat. No. 4,366,241, issued Dec. 28, 1982, herein incorporated by reference. (See especially columns 27 through 40.) Also, see Weng et al., U.S. Pat. No. 4,740,468, issued Apr. 26, 1988, herein incorporated by reference, especially at columns 2 and columns 6, 7, and 8.

The procedures for coupling enzymes to antibodies are well known in the art.

Reagents used for this procedure include glutaraldehyde, p-toluene diisocyanate, various carbodiimide reagents, p-benzoquinone, m-periodate, N, N*-o- phenylenedimaleimide, and the like (see, for example, J. H. Kennedy et al., Clin. Chim Acta lH: 1 (1976)).

Preferred signal generating agents are horseradish peroxidase and alkaline phosphatase.

Chemiluminescent labels are also applicable. See, for example, the labels listed in C. L. Maier, U.S. Pat. No. 4,104,029, issued Aug. 1, 1978, herein incorporated by reference.

The substrates for the catalytic systems include simple chromogens and fluorogens such as para-nitrophenyl phosphate (PNPP), β-D-glucose (plus possibly a suitable redox dye), homovanillic acid, o-dianisidine, bromocresol purple powder, 4- alkyl-umbelliferone, luminol, para-dimemylammoiophine, paramemoxylophine, and the like.

Depending on the nature of the label and catalytic signal producing system, one would observe the signal by irradiating with light and observing the level of fluorescence: providing for a catalyst system to produce a dye, fluorescence, or chemiluminescence, where the dye could be observed visually or in a spectrophotometer and the fluorescence could be observed visually or in a fluorometer; or in the case of chemiluminescence or a radioactive label, by employing a radiation counter. Where the appropriate equipment is not available, it will normally be desirable to have a chromophore produced that results in a visible color. Where sophisticated equipment is involved, any of the techniques is applicable. The term "solid phase" as used herein means common supports used in immunometric assays made from natural or synthetic materials. The solid phase support is insoluble in water and can be rigid or non-rigid. Among such supports are filter paper, the wells of microtiter plates, filtering devices (e.g., glass membranes), plastic beads (such as polystyrene beads), test tubes, strips, or (multiple) test wells made from polyethylene, polystyrene, polypropylene, nylon, nitrocellulose, glass microfibres, and the like. Also useful are particulate materials such as agarose, cross- linked dextran, and other polysaccharides.

The steps employed to remove the unbound components from the solid phase for the various assay formats can be performed by methods known in the art. Generally, a simple washing with buffer followed by filtration or aspiration is sufficient. After washing, it is sometimes appropriate, as with particulate supports, to centrifuge the support, to aspirate the washing liquid, add wash liquid again, and aspirate. For membrane and filters, additional washing with buffer may often be sufficient, preferably drawing the liquid through the membrane or filter by applying vacuum to the opposite side of the membrane or filter or contacting the opposite side of the filter or membrane with a liquid absorbing member that draws the liquid through, for instance, by capillary action.

Moderate temperatures, such as room temperature, are normally employed for carrying out the assay. Constant temperatures during the period of the measurement are generally required only if the assay is performed without comparison with a control sample. The temperatures for the determination will generally range from about 10°C to about-50°C, more usually from about 15°C to about -45°C.

In another embodiment according to the present invention, there are provided test kit(s) for the immunochemical detection of glyphosate, or salt(s) thereof, in a test sample. Invention test kit(s) comprise a solid phase, at least one invention anti-glyphosate antibody, which antibody is bound, or can be bound, to the solid phase. Invention test kit(s) may further comprise a labeled hapten conjugate that binds to the anti-glyphosate antibod(ies) to create a labeled anti-glyphosate antibody, and a linker selected from aspartic, glutamic, succinic, glutaric, adipic, N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic and poly-glutamic acids, and the like.

Invention test kit(s) may further be packaged in combination with predetermined amounts of reagents for use in assaying glyphosate. Where an enzyme is the label, the reagents can include substrate for the enzyme or the requisite precursors for the substrate, including any additional substrates, enzymes, and cofactors and any reaction partner of the enzymatic product required to provide the detectable chromophore or fluorophore. In addition, other additives, such as ancillary reagents, may be included, for example, stabilizers, buffers, and the like. The relative amounts of the various reagents may vary widely, to provide for concentrations in solution of the reagents which substantially optimize the sensitivity and specificity of the assay. The reagents can be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing the assay.

Invention test kit(s) are useful for determining the concentration of glyphosate or salt(s) thereof contained in such test samples taken from a variety of sources, e.g., a drinking water supply, an extract of an environmental specimen, an extract of a plant or soil specimen, an extract of a biological specimen, and the like. The detection sensitivity of the glyphosate in the sample is in the concentration range from about 100 ppm to about 0.5 ppb.

The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLE 1

All chemicals used in these Examples were reagent grade and commercially available from sources such as Sigma (St. Louis, MO), Chem Service (West Chester, PA), Amresco (Solon, OH), Molecular Devices (Sunnyvale, CA), Scripps (San Diego, CA), Corning (Kennebunck, MA), and Whatman (Clifton, NJ). Preparation of TG- and BSA-Glyphosate

Conjugates of glyphosate with TG or with BSA were prepared by activating the carboxylic groups of TG or BSA with l-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) followed by their coupling to the secondary amino group of glyphosate as follows. Fifty mg of EDC and 5 mg of sulfo-NHS

(Sulfo-N-hydroxysuccinimide) were added to 15 mg of TG pre-dissolved in 5 ml of 10 mM KH₂PO_4>pH 5.0. The reaction mixture was stirred for 2-3 minutes followed by the addition of 5 ml of a 2% solution of glyphosate in 0.2 M K₂HPO_4> pH 8.5. After the solution was stirred overnight, the conjugate (TG-glyphosate) was dialyzed exhaustively against PBS-7.4 (0.14 M NaCl, 10 mM K₂HPO_4>pH 7.4) or TBS-8

(tris-buffered saline, pH 8). The BSA-glyphosate conjugate was prepared using the same procedure. Both conjugates were stored at ≤-15°C. By this procedure the conjugate was formed by linkage to the carrier protein predominantly via the glutamic and aspartic acid residues.

EXAMPLE 2

Preparation of TG-Glyphosine

A conjugate of TG and glyphosine was prepared by activating the carboxylic group of glyphosine with EDC followed by its coupling to the amino groups of TG as follows. One hundred mg of EDC and 10 mg of Sulfo-NHS were added to 465 mg of glyphosine pre-dissolved in 2.5 ml of 10 mM KH₂PO_4>pH 5.0. After the reaction mixture was stirred for 2-3 minutes, the entire solution was transferred to a second reaction vessel containing 15 mg of TG pre-dissolved in 5 ml of 0.2 M K₂HPO_4> pH 8.5. The final reaction mixture was stirred overnight, followed by exhaustive dialysis against PBS at a pH of 7.4 or TBS at a pH of 8. The conjugate was stored at ≤ - 15°C. By this procedure the conjugate was formed by linkage to the carrier protein predominantly via the epsilon-amino groups of lysine residues. EXAMPLE 3

Production of Antibodies

Antibodies to TG-glyphosate conjugate and TG-glyphosine conjugate were produced in New Zealand white rabbits as follows. Rabbits were immunized with 0.5-1.0 mg of immunizing conjugate per rabbit per injection. The immunizing conjugate was emulsified with Complete Freund's Adjuvant for primary injections and with Incomplete Freund's Adjuvant for booster injections. Three or four booster injections were performed at monthly intervals to raise the desired titer to 10 to 50K. The rabbits were bled on 12±3 days following each booster injection. Antisera were monitored for titer and analyte specificity by capturing the relevant antibodies on ELISA plates coated with BSA-glyphosate conjugate. The captured antibodies were measured in a subsequent step by incubating the plates with an excess of goat anti-rabbit-IgG-horseradish peroxidase (GARIG-HRP).

EXAMPLE 4

Preparation of HRP-Glyphosate

An oligomeric form of HRP-glyphosate was prepared as follows. Thirty mg of HRP, pre-dissolved in 7.5 ml Buffer A (0.1 M sodium acetate, 0.15 M sodium chloride, pH 5.5), was stirred with 40 mg sodium m-periodate (25 mM) for 40 minutes in an ice-bath, followed by quenching with 0.3 ml of ethylene glycol for 5 minutes. The reaction mixture was dialyzed for approximately 4 hours against two liters of 1 :3 diluted Buffer A and the dialysate was then mixed with 2 ml of 0.5 M adipic acid dihydrazide in Buffer A. The reaction mixture was stirred at room temperature for about 1 hour and then at 2-6°C overnight, followed by extensive dialysis at 2-6°C against 1:3 diluted Buffer A. HRP-hydrazide prepared above was stored at 2-6°C. Next, a second portion of HRP was coupled to glyphosate as follows. Twenty mg EDC and 2 mg sulfo-NHS were added to 5 mg HRP (pre-dissolved in 2 ml of 10 mM KH₂PO₄, pH 5.0) and the reaction mixture was stirred for 2-3 minutes followed by the addition of 2 ml of a 2% solution of glyphosate in 0.2 M K₂HPO₄, pH 8.5. After stirring the reaction mixture for 1 hour at room temperature and then overnight at 2-6°C, the conjugate (HRP-glyphosate) was dialyzed exhaustively at 2-6°C against Buffer A. Finally, the HRP-glyphosate conjugate (5 mg) was further coupled to multiple copies of HRP-hydrazide (30 mg) as follows. HRP-glyphosate (5 mg in 5 ml Buffer A) was oxidized with 25 mM sodium m-periodate using essentially the same procedure as described above for the preparation of HRP-hydrazide. The dialysate from this reaction was combined with HRP-hydrazide prepared previously and the reaction mixture was stirred at room temperature for 30 minutes and then overnight at 2-6°C. The final conjugate was stored at < -15°C in the presence of 50 mM Trizma-8 (Sigma), 1% BSA, 0.01% thimerosal and 50% glycerol. The working aliquots of this conjugate can be stored at 2-6°C for at least 6 months.

EXAMPLE 5

Linker-Assisted ELISA for Glyphosate Using Antigen Coated Plates

On the basis of theoretical considerations, the antibodies raised against the TG-glyphosate and TG-glyphosine conjugates (i.e., the anti-TG-glyphosate and anti- TG-glyphosine antibodies) were expected to exhibit low affinity toward unbound glyphosate, a low molecular weight compound devoid of any rigid ring structure. In one ELISA format featuring antigen-coated microtiter plates, solid phase BSA- glyphosate competes against unbound glyphosate in the assay mixture, for binding to a limited amount of antibody in the assay mixture. In such assays, unbound glyphosate at concentrations lower than 2000 ng/ml is not able to compete effectively with the solid phase BSA-glyphosate, resulting in a relatively insensitive assay (See Figure 1). The antibody, however, shows high affinity toward the coating conjugate BSA-glyphosate, since the latter resembles the immunizing conjugates.

The present experiment was conducted to determine whether the affinity of the anti-TG-glyphosate and anti-TG-glyphosine antibodies prepared in Example 3 above could be enhanced by chemically modifying the analyte glyphosate in the sample to resemble the relevant epitopic structure of the immunogen and plate antigens used in a competition ELISA. Since the TG-glyphosate and TG-glyphosine conjugates were formed by linkage of glyphosate or glyphosine to the carrier proteins via amide or imide bonds involving glutamic acid, aspartic acid, and lysine residues of TG, it was contemplated that the affinity of the anti-TG-glyphosate and anti-TG-glyphosine antibodies for the analyte glyphosate in a sample could be enhanced by derivatization of analyte glyphosate with aspartic and glutamic acids, thereby incorporating an imide linkage into the analyte to resemble the relevant epitopic structure found on the immunogen and the plate antigen.

Initially, a mixture of aspartic and glutamic acids was tested for efficacy in affinity enhancement. The linkers, aspartic and glutamic acid, were activated with EDC for 2-5 minutes and then allowed to couple to glyphosate for about 30 min. via the secondary amine function of the glyphosate. A series of glyphosate-linker compounds were prepared by the same procedure using succinic, glutaric, adipic, N-acetyl-aspartic, N-acetyl glutamic, poly-aspartic, and poly-glutamic acids, and succinic and glutaric anhydrides as linkers. The anhydrides do not require pre- activation with a compound such as EDC.

It was discovered that the conjugation of glyphosate with the linker molecules resulted in a marked improvement in assay sensitivity. Highest enhancement in assay sensitivity was achieved with glutaric and succinic anhydrides.

The effect of pH on the assay was also investigated. As expected, the antibodies of the present invention bound derivatized glyphosate much more tightly at pH 9 than at pH 7. The binding was almost completely abolished at pH 5, most likely due to diminishing negative charge on the acidic groups of glyphosate. Furthermore, these antibodies exhibited virtually no cross-reactivity (less than 0.01%) toward arninomethylphosphonic acid (a major metabolite of glyphosate), thereby suggesting a critical role of the negative charges in antibody binding.

Based upon the foregoing, one embodiment of the invention, featuring antigen-coated plates, comprises the following steps:

1. Microtiter plates (Costar High Binding) are coated with BSA- glyphosate (Osborn reagent No. R0788), 0.2 ml/well, at 14 ng/ml water. After a coating period of 16-24 hours, the plates are over-coated with 1% BSA, 0.21 ml/well. Finally, the plates are rinsed twice with water, air-dried overnight and then stored at 2-6°C for up to at least two months.

2. 15x75 mm assay tubes are labeled with appropriate ID numbers and 10 μl of 0.5 M NaHCO₃ dispensed per tube.

3. 100 μl of each sample (standards, controls and unknowns) is dispensed per assay tube.

4. 20 microliters of a 5% solution of glutaric anhydride in DMSO are added per assay tube. Vortex and incubate the tubes at room temperature for approximately 30 minutes.

5. Anti-TG-glyphosine (Osborn reagent No. R0881) is diluted 1 :50,000 in IB-9 (50 mM Trizama-9®, 100 mM NaCl, 1% BSA, 0.1% Tween-20, 0.01% Thimerosal, 2.5 ppm Bromcresol Purple) and 700 μl of this solution is dispensed into each of the assay tubes. Vortex the tubes and then incubate them on a shaker for 10- 20 minutes.

6. Each sample is loaded into triplicate wells of a pre-coated plate (see step 1) and the plate is incubated on a shaker for approximately 1 hour.

7. The plate is washed once with EWB (0.85% NaCl, 0.005% Triton X- 100) using an automatic plate washer, GARIG-HRP (1:1000 dilution of Osborn reagent No. R0843 in IB-9) is added, and the plate is incubated on a shaker for approximately 45 minutes.

8. Finally, the plate is washed three times with EWB, and a tetramethylbenzidine-based HRP substrate is added (200 μl/well). The plate is incubated on a shaker for 10-20 minutes followed by the addition of 100 μl of Stop Solution/well (IN HCl). The plate is read at 450 nm using a computer-interfaced ELISA reader (V-max, Molecular Devices). Glyphosate concentration in the unknown samples is estimated from a concurrently-run standard curve. EXAMPLE 6

Linker-Assisted ELISA for Glyphosate Using Antibody-Coated Plates

Preparation of Pre-coated Plates

Antibody coated plates were prepared in batch using the following procedure. Microtiter plates (Costar High Binding) were coated overnight with protein A (2 μg/ml, 0.19 ml/well) in 0.2 M sodium bicarbonate. The plates were washed once with ELISA wash buffer (0.85% NaCl, 0.0005% Triton X-100). Rabbit anti-TG- glyphosate was diluted (e.g. 1:10,000, depending on the titer) with antibody incubation buffer (50 mM Trizma 9.1, 100 mM NaCl, 1% BSA, 0.1% Tween 20, 0.1% sodium azide), followed by dispensing of this solution (0.2 ml/well) into the wells of protein A coated plates. The plates were again incubated overnight and then washed twice with ELISA wash buffer containing 5% sucrose. Finally, the plates were air-dried overnight, sealed with plate sealing film, and stored at 2-6°C for up to at least three months. Finally, the plates were rinsed twice with deionized water, air-dried overnight and then stored at 2-6°C for up to at least two months.

Preparation of Buffer-coated Assay Tubes

A batch of assay tubes (15x75 mm polypropylene) were prepared by dispensing 20 μl of 0.5 M Trizma 9.1 (Sigma Chemicals) per tube and allowing complete drying (1-2 days at room temperature). These ready-to-use assay tubes can be stored at room temperature for at least 6 months.

Preparation of Succinylation Reagent

A stock solution of 2% succinic anhydride in dimethylsulfoxide (DMSO) was aliquoted into 2-ml portions. This reagent can be stored at 2-6°C for up to at least 3 months. The ELISA method

The presently prefened ELISA method of the present invention was carried out as follows: Buffer-coated assay tubes were labeled with appropriate ID numbers and 0.2 ml of each sample (standards, controls and unknowns) was dispensed per assay tube. To each tube was added 25 μl of succinylation reagent and the tubes were vortexed and then incubated at room temperature for approximately 20 minutes. Then the stock solution of HRP-glyphosate conjugate (prepared as in Example 4) was diluted 1:100 in IB-0.2 solution (0.2 M Trizma® 9.1, 1% BSA, 0.1% Tween®-20, 0.02T Thimerosal) and 0.6 ml of the dilute solution was added per assay tube.

Each sample was vortexed and loaded into triplicate wells (0.2 ml/well) of the pre-coated plate. The plate was sealed and incubated on a shaker for approximately 40 minutes, then washed three times with ELISA wash buffer, then 200 μl/well of a pre-formulated tetramethylbenzidine-hased HRP substrate was added. The plate was incubated on a shaker for approximately 10 minutes, the reaction was stopped by adding 100 μl well of stop solution (IN HCl), and the plate was read at 450 nm using a computer interfaced ELISA reader (Molecular Devices, Sunnyvale, CA). Glyphosate concentration in the unknown samples was estimated by comparison with a concurrently-run standard curve.

EXAMPLE 7 Glyphosate SPE Procedure

Solid phase extraction (SPE) for matrix cleanup or for concentration of samples was performed according to the following procedure.

Sample tubes (12x75 mm polypropylene tubes precoated with 20 μM Tris base), elution tubes (untreated 12 X 75 mm polypropylene tubes), and SPE columns (Whatman® SPE columns, SAX, 1 ml, 100 mg) were labeled with appropriate ID numbers, and the columns were preconditioned with 1 ml deionized water (di H₂O), using positive pressure to move the liquid through the column. Samples (standards, quality controls, and unknowns) were prepared by adding 2 ml of sample to sample tubes and vortexing. A 1 ml aliquot of each sample was loaded onto the appropriate preconditioned column, using positive pressure to move the Uquid through the column at a flow rate of approximately 1 m^rn-nute. The procedure was repeated using the remaining 1 ml of sample. Each column was then washed with 1 ml di H₂O, columns were transferred to appropriately labeled elution tubes, samples were eluted with 1 ml Eluent Solution (0.2 N HCl) using positive pressure to move the Uquid through the column at a flow rate of approximately 1 ml/minute, and tubes were vortexed.

The eluates were saved for glyphosate analysis by ELISA (Example 6).

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

WHAT IS CLAIMED IS:

1. A method for obtaining anti-glyphosate antibody(ies), said method comprising: preparing an immunogenic conjugate by covalently coupling glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, directly to a first carrier molecule, immunizing a susceptible host at variable intervals with said conjugate; and obtaining the antibodyries) from the host, wherein the coupling is under conditions selected to preserve the chemical identity of the at least two ionizable acidic groups in the derivative, if present.

2. The method according to claim 1 wherein the coupling between the glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, and the first carrier molecule is via an amide or imide linkage at the site of conjugation.

3. The method according to claim 2 wherein the glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, contributes a secondary amino or carboxyUc group, and the first carrier molecule contributes a carboxylic or primary amino group toward formation of the linkage.

4. The method according to claim 3 wherein the coupling involves an active ester.

5. The method according to claim 4 wherein the coupling involves a water soluble carbodiimide.

6. The method according to claim 5 wherein the coupling is carried out in two steps, comprising performing carbodiimide-mediated activation of carboxylic groups on the first carrier molecule under acidic pH conditions, and then reacting the activated carboxylic groups of the first carrier molecule, with the secondary amino groups of the hapten, under alkaline pH conditions.

7. The method according to claim 6 wherein the acidic pH is maintained between about 4 and about 6, and the alkaline pH is maintained between about 7.5 and 9.5.

8. The method according to claim 5 wherein the derivative is glyphosine, or a salt thereof, and the coupling is carried out in two steps, comprising performing carbodiimide-mediated activation of the derivative under acidic pH conditions, and then reacting the activated carboxylic group of the derivative, with the amino groups of the first carrier molecule under alkaline pH conditions.

9. The method according to claim 8 wherein the acidic pH is maintained between about 4 and about 6, and the alkaline pH is maintained between about 7.5 and 9.5.

10. The method according to claim 2 wherein the derivative is N-phosphonomethylglycine, or a structurally similar analog thereof.

11. The method according to claim 10 wherein the analog is N, N-bis(phosphonomethyl) glycine.

12. The method according to claim 10 wherein the analog is N,N-bis(phosphonomethyl) amine.

13. The method according to claim 10 wherein the analog is N-phosphonomethyliminodiacetic acid.

14. The method according to claim 10 wherein the analog is iminodiacetic acid.

15. The method according to claim 9 wherein the linkage is formed at the secondary amino group of the N-phosphonomethylglycine.

16. The method according to claim 10 wherein the linkage is formed at the carboxyl group of the N,N-bis(phosphonomethyl) glycine.

17. The method according to claim 11 wherein the linkage is formed at the secondary amino group of the N,N-bis(phosphonomethyl) amine.

18. The method according to claim 12 wherein the linkage is formed at one of the two carboxyl groups of the N-phosphonomethyUminodiacetic acid.

19. The method according to claim 13 wherein the linkage is formed at the secondary amino group of the iminodiacetic acid.

20. The method according to claim 1 wherein the first carrier molecule has a molecular weight in the range from about 100,000 to about 10,000,000.

21. The method according to claim 19 wherein the first carrier molecule is a protein selected from the group consisting of thyroglobulin, bovine serum albumin, human serum albumin, ovalbumin, and keyhole limpet hemocyanin.

22. The method according to claim 1 wherein the antibody is a polyclonal antibody or a functionally active fragment thereof.

23. The method according to claim 1 wherein the antibody is a monoclonal antibody or a functionally active fragment thereof.

24. The method according to claim 1 wherein the antibody is obtained using recombinant DNA technology.

25. The method according to claim 1 wherein said susceptible host is a rabbit.

26. The method according to claim 1 wherein the salt of glyphosate is a sodium salt.

27. An antibody obtained by the method according to claim 1.

28. A linker-assisted immunoassay method for the detection of an analyte in a test sample, said method comprising:

(a) reacting the test sample with a linker having an activated carboxylic group to obtain an analyte-linker conjugate in the test sample,

(b) contacting the reacted test sample with at least one anti-glyphosate antibody according to claim 27, (c) contacting the test sample containing the analyte-linker conjugate with a solid phase having immobilized thereon a solid phase coating conjugate comprising a second carrier molecule covalently coupled to glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, (d) removing unbound components from the solid phase, and

(e) detecting the presence of bound anti-glyphosate antibody, wherein the amount of bound antibody is inversely related to the amount of the glyphosate, or a salt thereof, in the test sample and wherein the second carrier molecule in the coating conjugate is not identical to the carrier molecule in the immunogenic conjugate used to obtain the anti-glyphosate antibody.

29. The method according to claim 28 wherein the linker has an activated carboxyUc group and the reacting is at a pH of about 7 to about 10.

30. The method according to claim 28 wherein the contacting of the reacted test sample with the anti-glyphosate antibody is in a buffer solution at a pH of 7 to 10.

31. The method according to claim 28 wherein the anti-glyphosate antibody is according to claim 19 wherein the first hapten is

N, N-bis(phosphonomethyl)glycine and the first carrier molecule is porcine thyroglobulin.

32. The method according to claim 28 wherein the anti-glyphosate antibody is according to claim 19 wherein the first hapten is N-phosphonomethylglycine and the first carrier molecule is porcine thyroglobulin.

33. The method according to claim 28 wherein the solid phase coating conjugate comprises N-phosphonomethylglycine covalently coupled to bovine serum albumin.

34. The method according to claim 28 wherein the solid phase coating conjugate comprises N, N-bis(phosphonomethyl)glycine covalently coupled to ovalbumin.

35. The method according to claim 28 wherein the linker is selected from the group consisting of aspartic, glutamic, succinic, glutaric, adipic, N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic and poly-glutamic acids.

36. The method according to claim 35 wherein the carboxylic group of the linker is activated by the water soluble carbodiimide method.

37. The method according to claim 36 wherein the linker is glutaric acid, or succinic acid, or a mixture thereof.

38. The method according to claim 28 wherein the linker is succinic anhydride, glutaric anhydride, or a mixture thereof.

39. The method according to claim 28 wherein the linker is covalently linked to the secondary amino group of glyphosate, thereby enhancing the affinity of the anti-glyphosate antibody for the glyphosate.

40. The method according to claim 28 wherein the anti-glyphosate antibody is conjugated to biotin and the detecting comprises contacting the anti-glyphosate antibody with an enzyme-labeled molecule that binds strongly to the biotin.

41. The method according to claim 28 further comprising binding to the anti-glyphosate antibody on the solid phase a second antibody conjugated to a signal- generating agent .

42. The method according to claim 41 wherein the signal-generating agent is selected from the group consisting of enzymes, radioisotopes, chemiluminescent and fluorescent labels, colored microbeads, and colloidal gold.

43. The method according to claim 41 wherein the signal-generating agent is horseradish peroxidase or alkaline phosphatase.

44. The method according to claim 28 wherein the solid phase is a microtiter well, a tube, or a strip.

45. A method for the immunochemical detection of glyphosate, or a salt thereof, in a test sample, said method comprising using an antibody according to claim 1 in an immunoassay format.

46. The method according to claim 45 wherein the immunoassay is a competitive homogeneous immunoassay.

47. The method according to claim 45 wherein the immunoassay is a direct competitive ELISA.

48. A test kit for the immunochemical detection of glyphosate, or a salt thereof, in a test sample, wherein the test kit comprises an anti-glyphosate antibody according to claim 28, a solid phase, and a coating conjugate comprising glyphosate, a derivative containing at least two ionizable acidic groups, or a salt thereof, covalently bound to a second carrier molecule different than the first carrier molecule, which carrier molecule is bound, or can be bound, to the solid phase.

49. A test kit according to claim 48 wherein the test kit further comprises a labeled substance that binds to the anti-glyphosate antibody to create a labeled anti-glyphosate antibody, and a linker selected from the group consisting of aspartic, glutamic, succinic, glutaric, adipic, N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic and poly-glutamic acids.

50. The test kit according to claim 48 wherein the solid phase coating conjugate comprises N-phosphonomethylglycine covalently coupled to bovine serum albumin.

51. The test kit according to claim 48 wherein the solid phase coating conjugate comprises N, N-bis(phosphonomethyl)glycine covalently coupled to ovalbumin.

52. The test kit according to claim 48 wherein the test sample is from a drinking water supply.

53. The test kit according to claim 48 wherein the test sample is a sample extract of an environmental specimen.

54. The test kit according to claim 48 wherein the test sample is a sample extract of a plant or soil specimen.

55. The test kit according to claim 48 wherein the test sample is a sample extract of a biological specimen.

56. The test kit according to claim 48 wherein the detection of the glyphosate in the sample is in the concentration range from about 100 ppm to about 0.5 ppb.