WO1991005259A1

WO1991005259A1 - Improved method for detecting pesticides at the picogram level

Info

Publication number: WO1991005259A1
Application number: PCT/US1990/005424
Authority: WO
Inventors: Johanne C. Strahan
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1989-10-06
Filing date: 1990-09-27
Publication date: 1991-04-18
Also published as: JPH05501156A; CA2067342A1; AU6627390A; EP0496804A1

Abstract

An improved antigen-capture enzyme-linked immunosorbent assay, for measuring the presence of a target pesticide in a medium, comprising the steps of: (a) forming a complex of the pesticide in the medium with an excess of a first antibody of known titer; (b) binding the free antibody from Step (a) to a coating conjugate that is bound to a solid phase; (c) binding a signal-generating labeled antibody to the antibody-coating conjugate complex of Step (b); and (d) determining the amount of pesticide in the medium by comparing the signal generated by the labeled antibody of Step (c) to the signal generated by running the above steps with a sample containing no pesticide and one or more samples containing known concentrations of pesticide; wherein the improvement comprises: (i) diluting the titer of the first antibody employed in Step (a) such that the concentration of pesticide needed to reduce the signal generated by the label in Step (d) by 50 % is less than one-half the concentration required without the dilution; and (ii) increasing the titer of the labeled antibody employed in Step (c) so that the total time necessary to run the assay is about the same as a normal assay without the dilution of (i); thereby (iii) increasing by a factor of at least two the sensitivity of the assay to detect target pesticide.

Description

TITLE IMPROVED METHOD FOR DETECTING PESTICIDES AT THE PICOGRAM LEVEL

Background of the Invention A variety of enzyme-linked immunosorbent assay (ELISA) formats have been employed to detect pesticides. A comprehensive review of the various assay formats may be found in "Practice and Theory of Immunoassay" by Tijssen, Vol. 15, 1985, Elsevier. And a summary of assays for pesticides was reported by Hammock et al., Pestic. Sci., 1989, 26, 303 to 317. The reported limits of detection vary widely as a result of the pesticide being measured, the assay format and the actual protocal. Claims for picogram levels of detection commonly involve a concentration step and commonly employ an immobilized antibody format.

An immobilized antigen format and reduction in concentration of antibody is a strategy for increasing assay sensitivity but the art teaches that the reliability of the measurement decreases as the antibody concentration decreases.

The earliest report of an ELISA assay for a sulfonylurea, chlorsulfuron, used the immobilized antigen format and a standard enzyme label, alkaline phosphatase, and reported detection at the nanogram/ml level: Kelley et al., _________________

Chem.. 1985, 33, pages 962 to 965.

Subsequently, chlorsulfuron detection was claimed at 10 picogram/ml minimum level of detection in the immobilized antigen format, by J. Sharp at the Eastern Analytical Symposium on October 7, 1988. Summary of the Invention This invention concerns an improved antigen-capture enzyme-linked immunosorbent assay, for measuring the presence of a target pesticide or derivative thereof (referred to hereafter as "pesticide" or "analyte") in a medium, comprising the steps of: (a) forming a complex of the pesticide in the medium with an excess of a first antibody of known titer;

(b) binding the free antibody from Step (a) to a coating conjugate that is bound to a solid phase; (c) binding a signal-generating labeled antibody to the antibody-coating conjugate complex of Step (b); and

(d) determining the amount of pesticide in the medium by comparing the signal generated by the labeled antibody of Step (c) to the signal generated by running the above steps with a sample containing no pesticide and one or more samples containing known concentrations of pesticide; wherein the improvement comprises: (i) diluting the titer of the first antibody employed in Step (a) such that the concentration of pesticide needed to reduce the signal generated by the label in Step (d) by 50% is less than one-half the concentration required without the dilution; and (ϋ) increasing the titer of the labeled antibody employed in Step (c) so that the total time necessary to run the assay is about the same as a normal assay without the dilution of (i); thereby (iii) increasing by a factor of at least two the sensitivity of the assay to detect target pesticide. By "coating conjugate" is meant a hapten chemically conjugated to a protein, sometimes called "coating antigen".

The present invention provides a method for rapid, sensitive and accurate measurement of picogram/ml concentrations of a pesticide or derivative in water, or in an aqueous extract of soil, food or crops, wherein the unprecedented sensitivity is obtained by dilution of the antigen-specific antibody relative to standard ELISA conditions. The art teaches reducing the amount of antibody to increase the sensitivity of the assay but also teaches that the increase in sensitivity may be accompanied by a decrease in precision. In addition, the technical difficulty of detecting very low concentrations of antibodies in a timely fashion to prevent time dependent changes precludes the practical application of this technique to obtain high sensitivity. However, an interrelated aspect of the present invention provides for an increase in concentration of the labeled antibody to restore detection to a standard assay time frame. And the precision, contrary to the teaching, does not decrease but may improve. The resulting sensitivity is in the 1 to 100 picogram/ml range in an aqueous medium. Samples containing higher concentrations may be diluted into this range, which offers the advantage of significant reduction in errors introduced by too-high concentrations of unknown materials in the medium; that is, a reduction in matrix effects.

The method as outlined above may be used in either a manual kit format or in an automated system. Preferred first antibody is rabbit polyclonal antibody produced by methods described hereafter. Preferred labeled antibody-enzyme conjugate is alkaline phosphatase-goat anti-rabbit IgG. Preferred protein-antigen conjugates (coating conjugates) are described hereafter. Preferred as a solid phase support is a 96-well microtiter plate.

Details of the Invention

The ELISA Technique

The particular ELISA employed in this invention makes use of antigen capture in the following steps. The selected antibody is contacted with a test sample containing the pesticide, whereby a fraction of said antibody forms a complex with the pesticide and the balance remains free. The free antibody is then separated from the reaction mixture by contacting the reaction mixture with a solid phase comprising the antigen or an analog of the antigen immobilized on a solid phase. The reaction mixture is then removed from the solid phase by washing, and the solid phase is contacted with a labeled second antibody then which reacts with the bound first antibody. The unbound labeled second antibody is removed by washing and the amount of first antibody-labeled second antibody complex that has been formed is measured using the label. The amount of pesticide in the sample is then determined, photometrically, or by any other art-recognized method, by comparison with a standard curve derived by running the assay with several known concentrations of pesticide.

Vallejo et al., J. Aαric. Food Chem.. 1982, 2SL, pages 572 to 580 studied different hapten structures for parathion. He concluded that "the determinant groups of the small molecule must be preserved" and that "the hapten's determinant groups must not be masked" for antibody production. The format most commonly used for pesticide immunoassays has utilized a solid phase with hapten bound to it (coating conjugate) for capture of antibodies not bound to free compound. The quantification of the captured antibody is used to determine the original concentration of compound in the original aqueous sample.

Hapten structures were further explored by ie et al., J. Aσric. Food Chem., 1984, _\__, pages 1294 to 1301 for method development for an assay for diflubenzuron. Their main purpose was to show that sensitive assays could be achieved by using a coating conjugate of different structure than the immunizing hapten. In particular, they demonstrated a different linker arm and different position of linker arm gave more sensitive assays. In addition, some functional group changes in the coating conjugate structure rendered assays which detected a class of compounds. Thus, they gained some sensitivity by shifting linker arm and gained a desired lack of specificity by changing functional groups.

Van Emon et al., "Analytical Methods for Pesticides and Plant Growth Regulators", Vol. XVII, 1989, pages 217 to 263 state that "the point of attachment of hapten to protein should occur away from any suspected antigenic determinants" to insure proper antigenic response for developing specific antibodies. They also state that to develop a compound class specific assay the hapten structure for immunization is important, particularly preserving the common antigenic determinants of the related compounds.

In the instant invention, the hapten in the coating conjugate is selected so that it is structurally similar both to the pesticide to be analyzed and to the hapten in the conjugate against which antibody was raised. The carboxylate moiety is used for attachment of the hapten to the coating protein. The best coating protein/hapten combination is determined empirically based on maximizing affinity for the first antibody.

Examples of haptens that are useful in this invention are as follows:

H₃C ^CH3

H,C^ CH₃

15

25

35

15

25

35

15

25

35 Examples of proteins that are useful in the method of this invention include keyhole limpet hemocyanin, ovalbumin, globulins such as pumpkin seed or marijuana seed, and serum albumin from cows, rabbits or mice.

The solid phase component of Step (b) is chosen for its characteristics of efficient immobilization of the coating conjugate, for its non-specific binding characteristics, and its ease of use in the separation of free antibody from bound antibody.

The solid phase material can be fabricated from any number of synthetic materials which can take up the protein-antigen conjugate in a reproducible manner. Preferably, the solid phase material can be fabricated from non-porous metal oxides, polymeric materials such as agarose, polystyrene, polyacrylamide, their derivatives or mixtures thereof. It can be present pre-formed as a particle, bead, a microplate, dip-stick, filter paper, tube, magnetic particle or a matrix having a density greater than water. The solid phase material can be delivered to the reaction vessel as a dry powder, wet slurry, tablet, capsule or as a pre-formed distinct shape.

Immobilized on the solid phase material is an optimized concentration of coating conjugate where the hapten is structurally similar to the unknown being analyzed, and is capable of binding the free first antibody in the reaction mixture which consists of the target pesticide and the pesticide-specific polyclonal first antibody. The reaction mixture and the treated solid phase material become separated into a solid phase containing the coating conjugate bound with any excess first antibody which will subsequently be quantified, and a liquid phase containing the soluble antibody-pesticide complex which is removed from the solid phase by washing.

The immobilization of the coating conjugate on the solid phase is preferentially by passive adsorption of the protein but can be by covalent bonding either directly or through a spacer arm, or by an avidin-biotin binding where the solid phase is avidinylated or biotinylated and the protein is biotinylated or avidinylated. These methods are known in the art. See, Wilchek et al., Analytical Biochem. 171. 1988, pages 1 to 32. An important aspect of this invention is that the dilution of the antiserum reduces the antibody concentration to produce an affinity driven reaction and the avidity of the polyclonal antiserum contributes to the binding of the free antibody in the reaction mixture to the hapten on the solid phase. In one preferred embodiment, the hapten conjugate is an analogue of the unknown suspect pesticide, which can provide for an additional difference in affinity and is complementary to the "avidity" effect. The term avidity refers to the sum of the affinity constants of the polyclonal antibody.

Subsequent to removal of the liquid phase by washing, the bound second antibody is measured via its attached label. The second antibody concentration is inversely proportional to the concentration of the unknown in the sample, determined by comparison to a standard curve.

Generally, this is accomplished by introducing a labeled second antibody which is directed against the Fc portion of the bound first antibody. Typical labels include enzymes, radioisotopes, chromophores, fluorophores or any substance capable of generating a detectable signal, either alone or in combination with other reagents. Procedures and methods for labeling and identifying the labeled complexes are known in the art of diagnostic immunoassay and are generally discussed in Miles et al., "Labelled Antibodies and Immunological Assay Systems", Nature. 219_, pages 187 to 189 (1968) and U.S. 3,654,090. The preferred label in the instant invention is an anti-rabbit antibody-alkaline phosphatase conjugate and a p-nitrophenyl phosphate substrate for quantitation. The enzyme label is preferred due to the availability of sensitive chromogenic substrates and simple instrumentation to quantitate results.

Sulfonylureas whose presence can be detected and measured by the method of this invention include:

2-chloro-N-[(4-methoxy-6-methyl-l,3,5-triazin-2- yl)amino]carbonyl]benzenesulfonamide Chlorsulfuron methyl2-[ [ [ [ (4, 6-dimethyl-2-pyrimidinyl)amino]- carbonyl]amino]suIfonyl]benzoate Sulfometuron methyl methyl-2-[ [ [ [(4-methoxy-6-methyl-l,3,5-triazin-2- yl)amino]carbonyl]amino]sulfonyl]benzoate Metsulfuron methyl 2-[ [N-(4-methoxy-6-methyl-l,3,5-triazin-2-yl)-N- methylamino]carbonyl]amino]sulfonyl]benzoic acid, methyl ester Express (TM) ethyl 2-[ [ [ [ (4-chloro-6-methoxy-2-pyrimidinyl) , amino]carbonyl]amino]sulfonyl]benzoate Chlorimuron ethyl 2- [ [ ( 4-ethoxy-6-methylamino-l , 3 , 5-triazin-2- yl)aminocarbonyl]aminosulfonyl]benzoic acid, methyl ester Muster (TM) 2-[[(4,6-dimethoxy-l,3,5-triazin-2-yl)aminocarbonyl]- aminosulfonyl]-4-(2,2,2-trifluoroethoxy)benzoic acid, ethyl ester 4-chloro-2-[ [(4-methoxy_r6-methyl-l,3,5-triazin-2- yl)aminocarbonyl]aminosulfonyl]benzoic acid, isopropyl ester 3-[ [ [[(4-methoxy-6-methyl-l,3,5-triazin-2-yl)amino]- carbonyl]amino]sulfonyl]-2-thiophene carboxylic acid, methyl ester Thiameturon methyl methyl 2-[ [ [[(4,6-dimethoxy-2-pyrimidinyl)amino]- carbonyl]amino]sulfonyl]methylbenzoate Bensulfuron methyl 2-[ [(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]- aminosulfonyl]N,N-dimethy1-3-pyridinecarboxamide 2-[[(4,6-dimethoxypyrimidin-2-yl))aminocarbonyl]- aminosulfonyl]-3-pyridinecarboxylic acid, methyl ester N-[(4, 6-dimethoxypyrimidin-2-yl))aminocarbonyl]-3- (ethylsulfonyl)-2-pyridinesulfonamide N-[(4,6-dimethoxypyrimidin-2-yl))aminocarbonyl]- 2,3-dihydro-2-methyl-benzo(b)thiophene-7- sulfonamide, 1,1 dioxide 2-[[ [ [(4, 6-bis(difluoromethoxy)-2-pyrimidinyl]- amino]carbonyl]amino]sulfonyl]benzoic acid, methyl ester ethyl 5-[3-( ,6-dimethoxypyrimidin-2-yl)ureido- sulfonyl]-l-methylpyrazole-4-carboxylate N-[(6-methoxy-4-methyl-1,3,5-triazin-2-yl)amino¬ carbonyl]-2-(2-chloroethoxy)benzene sulfonamide N-[(4,6-dimethoxy-l,3, 5-triazin-2-yl)amino-carbonyl] 2-(2-methoxyethoxy)benzenesulfonamide N-[ (4, 6-dimethoxyρyrimidin-2-yl)-amino]carbonyl]- 3-trifluoromethyl-2-pyridinesulfonamide.

Other types of herbicides that can be advantageously measured by employing the improved assay of this invention are given below:

Common Name Chemical Name

acetochlor 2-chloro-N-(ethoxymethyl)-

N(2-ethyl-6-methylphenyl)- acetamide acifluorfen 5-[2-chloro-4-(rrifluoromethyl)- phenoxy]-2-nitrobenzoic acid acrolein 2-propenal alachlor 2-chloro-N-(2,6-diethylphenyl)-

N(methoxymethyl)acetamide ametryn N-ethyl-N'-(l-methylethyl)-6-

(methylthio)-l,3,5-triazine-

2,4diamine amitrole 1H-1,2,4-triazol-3-amine AMS ammonium sulfamate asulam methyl [(4-aminophenyl)sulfonyl]- carbamate atrazine 6-chloro-N-ethyl- ^•-(1-me hyl- e h l)l,3,5-triazine-2,4- diamine barban 4-chloro-2-butynyl 3-chloro- carbamate benefin N-butyl-N-ethyl-2, 6-dinitro-4-

(trifluoromethyl)benzenamine bensulide 0,0-bis(l-methylethyl) S-

[2[ (phenylsulfonyl)amino]- ethyl]phosphorodithioate bentazon 3-(l-methylethyl)-(lH)-2,l,3- benzothiadiazin-4(3H)-one,

2,2-dioxide benzofluor N-[4-(ethylthio)-2-(trifluoro- methy1)pheny1]methane- sulfonamide benzoylprop N-benzoyl-N-(3,4-dichlorophenyl)-

DLalanine bifenox methyl 5-(2,4-dichloro- phenoxy)-2nitrobenzoate bromacil 5-bromo-6-methyl-3-(l-methylpro- . pyl)2,4(lH,3H)pyrimidinedione bromoxynil 3,5-dibromo-4-hydrσxybenzonitrile butachlor N-(butoxymethyl)-2-chloro-N-

_*

(2,δdiethylphenyl)acetamide buthidazole 3-[5-(l,l-dimethylethyl)-l,3,4- thiadiazol-2-yl]-4-hydroxy-l- methyl-2imidazolidinone butralin 4-(1,1-dimethylethy1)-N-(1- methylpropyl)-2,6-dinitro- benzenamine butylate S-ethyl bis(2-methylpropyl)car- bamothioate cacodylic dimethyl arεinic oxide acid CDAA 2-chloro-N,N-di-2-proρenyl- acetamide

CDEC 2-chloroallyl diethyldi- thiocarbamate chloramben 3-amino-2,5-dichlorobenzoic acid chlorbromuron 3-(4-bromo-3-chlorophenyl)-l- methoxy-lmethylurea chlorimuron 2-tC[[(4-chloro-6-methoxy-2- ethyl pyrimiethyldinyl)ethylamino]- carbonyl]amino]sulfonyl]benzoic acid, ethyl ester chloroxuron N'-[ -(4-chlorophenox )phen 1]-

N,Ndimethylurea chlorpropham 1-methylethyl 3-chIorophenyl- carbamate chlortoluron N'-(3-chloro-4-methylphenyl)-

N,Ndimethylurea cinmethylin exo-l-methyl-4-(1-methylethyl)-2- [(2methylphenyl)methoxy]-7- oxabicyclo[2.2.1]heptane clethodim (E,E)-(+)-2-[l-[[(3-chloro-2- propenyl)oxy]imino]propyl]-5- [2-(ethylthio)propyl]-3- hydroxy-2-cyclohexen-l-one clomazone 2-[(2-chlorophenyl)methyl]-4,4- dimethyl3-isoxazolidinone cloproxydim (E,E)-2-[l-[ [ (3-chloro-2-pro- ρenyl)oxy)imino]butyl]-5- [2-(ethylthio)propyl]3-hydroxy- 2-cyclohexen-l-one clopyralid 3, 6-dichloro-2-ρyridinecar- boxylic acid

CMA calcium salt of MAA cyanazine 2-[ [4-chloro-6-(ethylamino)- l,3,5-triazin-2-yl]amino]-2- methylpropanenitrile cycloate S-ethyl cyclohexylethylcar- bamothioate cycluron 3-cyclooctyl-l,1-dimethylurea cyperquat l-methyl-4-phenylρyridinium cyprazine 2-chloro-4-(eyelopropy1amino)-

6-(isopropylamino)-s-triazine cyprazole N-[5-(2-chloro-l, 1-dimethyl- ethy1)-1,3,4thiadiazol-2- yl]cyclopropanecarboxamide cypromid 3' ,4'-dichlorocyclopropane- carboxanilide dalapon 2,2-dichloropropanoic acid dazomet tetrahydro-3,5-dimethyl-2H-

1,3,5-thiadiazine-2-thione

DCPA dimethyl 2,3,5,6-tetrachloro-

1,4-benzene-dicarboxylate desmediphan ethyl [3-[ [(phenylaminoj- carbonyljoxyjphenyljcarba ate desmetryn 2-(isopropylamino)-4-(methyl- amino)-6(methylthio)-s- triazine diallate S-(2,3-dichloro-2-propenyl)- bis(lmethylethyl)carbamothioate dicamba 3,6-dichloro-2-methoxybenzoic acid dichlobenil 2,6-dichlorobenzonitrile dichlorprop (+)-2-(2,4-dichlorophenoxy)- propanoic acid dichlofop (+)-2-[4-(2,4-dichlorophenoxy)- phenoxy]proρanoic acid diethatyl N-(chloroacetyl)-N-(2 ,6-diethyl- phenyl)glycine difenzoquat 1,2-dimethyl-3,5-diphenyl-lH- pyrazolium dinitramine N3 , 3-diethyl-2 ,4-dinitro-6-

(trifluoromethyl)-l,3- benzenediamine dinoseb 2-(l-methylpropyl)- ,6-dinitro- phenol diphenamid N,N-dimethyl-a-phenylbenzene- acetamide dipropetryn 6-(ethylthio)-N,N'-bis(1-methyl¬ ethyl)1,3,5-triazine-2,4- diamine

6,7-dihydrodipyrido[l,2-a:2 ' ,1'- c]pyrazinedium ion

N'-(3,4-dichlorophenyl)-N,N- dimethylurea

2-methyl-4, 6-dinitrophenol disodium salt of MAA

7-oxabicyclo[2.2.1]heptane-2,3- dicarboxylic acid

S-ethyl dipropylcarbamothioate

N-ethyl-N-(2-methyl-2-propenyl)- 2,6dinitro-4-(trifluoro- methyl) enzenamine ethofumesate (+)-2-ethoxy-2,3-dihydro-3,3- dimethyl5-benzofuranyl methanesulfonate fenac 2,3,6-trichlorobenzeneacetic acid fenoxaprop (+)-2-[4-[(6-chloro-2-benzoxa- zolyl)oxy] henoxy]propanoic acid fenuron ,N-dimethyl-N*-phenylurea fenuron TCA Salt of fenuron and TCA flamprop N-benzoyl-N-(3-chloro-4-fluoro- phenyl)DL-alanine fluazifop (+)-2-[4-[[5-(trifluoromethyl)-2- pyridinyl]oxy] henoxy]pro¬ panoic acid fluazifop-P (R)-2-[4-[[5-(trifluoromethyl)-2- pyridinyl]oxy]phenoxy]pro¬ panoic acid fluchloralin N-(2-chloroethyl)-2, 6-dinitro-N- propy14-(trifluoromethyl)- benzenamine fluometuron N,N-dimethyl-N'-[3-(trifluoro¬ methyl)phenyl]urea fluorochlor- 3-chloro-4-(chloromethyl)-l-[3- idone (trifluoromethyl)phenyl]- 2-pyrrolidinone fluorodifen p-nitrophenyl α,α,α-trifluoro-2- nitro-p-tolyl ether fluorogly- carboxymethyl 5-[2-chloro-4- cofen (trifluoromethyl)phenoxy]- 2-nitrobenzoate fluridone l-methyl-3-phenyl-5-[3-(tri- fluoromethyl) henyl]-4(IH)- pyridinone fluroxypyr acetic acid, 2-[[[4-amino- 3,5-dichloro-6-fluoro-2- pyridinyl]oxy] ]-, [ [methyl- heptyl]ester] fomesafen 5-[2-chloro-4-(trifluoromethyl)- phenoxy]N-(methylsulfonyl)- 2-nitrobenzamide fosamine ethyl hydrogen (aminocarbonyl)- phosphate glyphosate N-(phosphonomethyl)glycine haloxyfop 2-[4-[ [3-chloro-5-(trifluoro¬ methyl)-2-pyridinyl]oxy]- phenoxy]propanoic acid hexaflurate potassium hexafluoroarsenate hexazinone 3-eyelohexy1-6-(dimeth lamino)- 1-methyll,3,5-triazine- 2,4(lH,3H)-dione imazametha- 6-(4-isopropy1-4-methy1-5-oxo- benz 2-imidazolin-2-yl)- m-toluic acid, methyl ester and 6-(4-isopropyl4-methyl-5- oxo-2-imidazolin-2-yl)p- toluic acid, methyl ester imazapyr (+)-2-[4,5-dihydro-4-methyl-4-(l- methylethyl)-5-oxo-lH- imidazol-2-yl]-3pyridine- carboxylic acid imazaquin 2-[4,5-dihydro-4-methyl-4- (1-methylethyl)-5-oxo-lH- imidazol-2-yl]-3quinoline- carboxylic acid imazethapyr (+)-2-[4,5-dihydro-4-methyl-4-(l- methylethyl)-5-oxo-lH-imidazol- 2-yl]-5ethyl-3-pyridinecar- boxylic acid ioxynil 4-hydroxy-3,5-diiodobenzonitrile isopropalin 4-(l-methylethyl)-2,6-dinitro-

N,Ndipropylbenzenamine isoproturon N-(4-isopropylphenyl)-N' ,N'- dimethylurea isouron '-[5-(1,1-dimethylethy1)-3- isoxazolyl]N,N-dimethylurea isoxaben N-[3-(l-ethyl-l-methylpropyl)- 5isoxazolyl]-2, 6-dimethoxy- benzamide karbutilate 3-[ [(dimethylamino)carbonyl]- amino]phenyl-(l,1-dimethyl¬ ethyl)carbamate lactofen (+)-2-ethoxy-l-methyl-2-oxo- ethyl 5-[2chloro-4-(trifluoro¬¬ methyl)phenoxy]2-nitrobenzoate lenacil 3-cyclohexyl-6,7-dihydro-lH- cyclopentapyrimidine-

2,4(3H,5H)-dione linuron N'-(3, -dichloroρhenyl)-N- methoxy-Nmethylurea

MAA methylarsonic acid

MAMA monoammonium salt of MAA

MCPA (4_rchloro-2-methyIphenoxy)acetic acid

MCPB 4-(4-chloro-2- ethyIphenoxy)- butanoic acid mecoprop (+)-2-(4-chloro-2-methylphenoxy)- propanoic acid mef luidide N-[2,4-dimethyl-5-[[(trifluoro¬ methyl)sulfonyl]amino]phenyl]- acetamide methal- N-(2-methyl-2-propenyl)-2, 6- propalin dinitro-N-4-(tri- fluoromethyl)benzenamide methabenz- 1,3-dirnethyl-3-(2-benzothia- thiazuron zolyl)urea metham methylcarbamodithioic acid methazole 2-(3,4-dichlorophenyl)-4-methyl- l,2,4oxadiazolidine-3,5-dione methoxuron '-(3-chloro-4-methoxyphenyl)-

N,Ndimethylurea metolachlor 2-chloro-N-(2-ethyl-6-methyl- pheny1)- (2-methoxy-1- methylethyl)acetamide metribuzin 4-amino-6-(l,1-dimethylethyl)-

3-(methylthio)-l,2,4-triazin-

5(4H)-one

MH 1,2-dihydro-3, 6-pyridazinedione mo linate S-ethyl hexahydro-lH-azepine-

1-carbothioate monolinuron 3-(p-chlorophenyl)-1-methoxy-l- methylurea monuron '-(4-chlorophenyl)- ,N-dimethyl- urea monuron TCA Salt of monuron and TCA MSMA rnonosodium salt of MAA napropamide N,N-diethyl-2-(l-naphthalenyl- oxy)propanamide naptalam 2-[(l-naphthalenylamino)carbo- nyl]benzoic acid neburon l-butyl-3-(3, -dichlorophenyl)-

1-methylurea nitralin 4-(methylsulfonyl)-2, 6-dinitro-

N,Ndipropylaniline nitrofen 2,4-dichloro-l-(4-nitrophenoxy)- benzene nitrofluorfen 2-chloro-l-(4-nitrophenoxy)-4-

(trifluoromethyl)benzene norea ,N-dimethyl-N'-(octahydro-4,7- methanolH-inden-5-yl)urea

3aα,4α,5α,7α,7aα-isomer norflurazon 4-chloro-5-(methylamino)-2-[3-

(trifluoromethyl)phenyl]-

3(2H)pyridazinone oryzalin 4-(dipropylamino)-3,5-dinitro- benzenesulfonamide oxadiazon 3-[2,4-dichloro-5-(l-methyl- ethoxy)phenyl]-5-(1,1- dimethylethyl)1,3,4-oxa- diazol-2(3H)-one oxyfluorfen 2-chloro-l-(3-ethoxy-4-nitro¬ phenoxy)-4-(trifluoromethyl)- benzene paraquat 1,1'-dimethyl-4,4 '-dipyridin- ium ion pebulate S-propyl butylethylcarbamothioate pendimethalin N-(l-ethylρropyl)-3,4-dimethyl- 2,δdinitrobenzenamine perfluidone 1,1,1-tri luoro-N-[2-methyl-4- (phenylsuIfonyl)phenyl]methane- sulfonamide phenmedipham 3-[(methoxycarbonyl)amino]phenyl- (3methylphenyl)carbamate picloram 4-amino-3,5, 6-trichloro-2- pyridinecarboxylic acid

PPG-1013 5-[2-chloro-4-(trifluoromethyl)- phenoxy]-2-nitroacetophenone oxime-O-acetic acid, methyl ester procyazme 2-[[4-chloro-6-(cyclopropyl- amino)-l,3,5triazine-2- yl]amino]-2-methylpropane- nitrile profluralin N-(cyclopropylmethyl)-2,6- dinitro-Npropyl-4-(tri- fluoromethyl)benzenamine prometon 6-methoxy-N,N'-bis(1-methyl¬ ethyl)-1,3,5triazine-2,4- diamine prometryn N,N"-bis(1-methylethyl)-6-

( ethylthio)1,3,5-triazine-2,4- diamine pronamide 3,5-dichloro-N-(l,l-dimethyl-2- propynyl)benzamide propachlor 2-chloro-N-(1-methylethyl)-N- phenylacetamide propanil N-(3,4-dichlorophenyl)propanamide propazine 6-chloro-N,N*-bis(1-methylethyl)-

1,3,5-triazine-2,4-diamine propham 1-methylethyl phenylcarbamate prosulfalin N-[ [4-(dipropylamino)-3,5- dinitrophenyl]sulfonyl]-S,S- dimethylsulfilimine prynachlor 2-chloro-N-(l-methyl-2-pro- pynyl)acetanilide pyrazon 5-amino-4-chloro-2-pheny1-

3(2H)pyridazinone quizalofop (+)-2-[4-[(6-chloro-2- quinoxalinyl)ethyloxy]- phenoxy]propanoic acid, ethyl ester secbumeton N-ethyl-6-methoxy-N'-(l- methylpropyl)1,3,5-triazine- 2,4-diamine sethoxydim 2-[1-(ethoxyimino)butyl]-5- [2-(ethylthio)proρyl]-3- hydroxy-2-cyclohexen-l-one siduron N-(2-methylcyclohexyl)-N'- phenylurea simazme 6-chloro-N,N'-diethyl-1,3,5- triazine2,4-diamine sulfometuron 2-[ [ [ [(4,6-dimethyl-2-pyrimi- dinyl)methylamino]carbonyl]- amino]sulfonyl]benzoic acid, methyl ester

TCA trichloroacetic acid tebuthiuron N-[5-(l,l-dimethylethyl)-l,3,4- thiadiazol-2-yl]-N,N'- dimethylurea terbacil 5-chloro-3-(l,1-dimethylethyl)-

6-methyl-2,4(lH,3H)- pyrimidinedione terbuchlor N-(butoxymethy1)-2-chloro-N-[2-

_.(1,ldimethylethyl)-6-methyl- phenyl]acetamide terbuthyl- 2-(tert-butylamino)-4-chloro-6- azine (ethylamino)-s-triazine terbutol 2,6-di-tert-butyl-p-tolyl methylcarbamate terbutryn N-(1,1-dimethylethyl)-N^•-ethyl-

6(methylthio)-l,3,5-triazine-

2,4-diamine thiobencarb S-[(4-chlorophenyl)methyl] diethylcarbamothioate triallate S-(2,3,3-trichloro-2-propenyl)- bis(lmethylethyl)carbamothioate triclopyr [(3,5,6-trichloro-2-pyridinyl)- oxy]acetic acid tridiphane 2-(3,5-dichlorophenyl)-2-

(2,2,2trichloroethyl)oxirane 2 ,6-dinitro-N,N-dipropyl-4-

(trifluoromethyl)benzenamine l-(p-chlorophenyl)-2,3,3-tri- methylpseudourea (2,4-dichloroρhenoxy)acetic acid 4-(2,4-dichlorophenoxy)butanoic acid S-propyl dipropylcarbamothioate

2-chloro-N-(2,3-dimethylphenyl)-

N(1-methylethyl)acetamide Fungicides methyl 2-benzimidazolecarbamate (carbendazim) tetramethylthiuram disulfide (thiuram) n-dodecylguanidine acetate (dodine) manganese ethylenebisdithiocarbamate (maneb) 1,4-dichloro-2,5-dimethoxybenzene (chloroneb) methyl 1-(butylearbamoyl)-2-benzimidazolecarbamate (benomyl)

2-cyano-N-ethylearbamoy1-2-methoxyiminoacetamide

(cymoxanil) N-trichloromethylthiotetrahydrophthalamide (ca tan) N-trichloromethylthiophthalimide (folpet) dimethyl 4,4 '-(o-phenylene)bis(3-thioallophanate) (thiophanate-methyl) 2-(thiazol-4-yl)benzimidazole (thiabendazole) aluminum tris(0-ethyl phosphonate) (phosethyl aluminum) tetrachloroisophthalonitrile (chlorothalonil) 2, 6-dichloro-4-nitroaniline (dichloran) N-(2, 6-dimethylphenyl)-N-(methoxyacetyl)alanine methyl ester (metalaxyl) cis-N-[l,1,2,2-tetrachloroethyl)thio]cyclohex-4- ene-l,2-dicarbioximide (captafol)

3-(3,5-dichlorophenyl)-N-(1-methylethyl)-2,4- dioxo-1-imidazolidine carboxamide (iprodione) 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl-2,4- oxazolidinedione (vinclozolin) kasugamycin

O-ethyl-S,S-diphenylphosphorodithioate (edifenphos) 4-(3-(4-(l,l-dimethyl-ethyl)phenyl)-2-methyl) ropyl-

2,6-dimethylmorpholine (fenpropimorph) 4-(3-4(1,1-dimethy1-ethy1)phenyl)-2-methyl)propylpi peridine (fenpropidine) l-(4-chlorophenoxy)-3,3-dimethyl-l-(lH-l,2,4-triazol- l-yl)butanone (Bayleton, triadimefon) 3-(4-chlorophenoxy)-α-(1,1-dimethylethyl)-1-H-l, 2 , 4- triazol-1-ethanol (Baytan, triadimenol) 2-(4-chlσrophenyl)-2-(lH-l,2,4-triazol-l-ylmethyl)- hexanenitrile (Systhane®, myclobutanil) Folicur®

(tebuconazol) 3-chloro-4-[4-methyl-2-(1H-1,2,4-triazol)-l-ylmethyl)-

1,3-dioxolan-2-yl]phenyl-4-chlorophenyl ether (Score®) l-[2-(2,4-dichlorophenyl)pentyl]1H-1,2,4-triazole

(Topas®, penconazole) +-α-(2-fluorophenyl-α-(4-fluorophenyl)-lH-

1,2,4-triazole-l-ethanol (Impact®, flutriafol) l-[ [bis(4-fluorophenyl)methylsilyl)methyl]-lH-l,2,4- triazole (Nustar®, flusilazol) 1-N-propyl-N-[2(2,4,6-trichlorophenoxy)ethyl]- carbamoylimidazole (Sportak®, prochloraz) l-[ [2-(2,4-dichlorophenyl)-4-propy1-1,3-dioxolan-

2-yl]methyl]-1H-1,2,4-triazole (Tilt®, propiconazole) (+/-)-α-butyl-α-(2,4-dichloroρhenyl)-lH-l,2,4- triazole-1-ethanol (Anvil®, hexaconazole) α-(2-chlorophenyl)-α-(4-chlorophenyl)-5-pyridine- methanol (Rubigan®, fenarimol) methyl N-(2,6-dimethylphenyl)-N-(2-furanylcarbonyl)-

DL-alaninate (Fongarid®, furalaxyl)

Bactericides streptomycin sulfate oxytetracycline (dihydrate) Acaricides trans-5-(4-chloroρhenyl)-N-cyclohexyl-4-methyl-2- oxo-3-thiazolidinecarboxamide (Savey®, hexythiazox) senecioic acid, ester with 2-sec-butyl-4,.6-dinitro- phenol (binapacryl) 6-methyl-l,3-dithiolo[2,3-B]quinonolin-2-one (Morestan®, oxythioquinox) 2,2,2-trichloro-l,l-bis(4-chlorophenyl)ethanol

(Kelthane®, dicofol) bis(pentachloro-2,4-cyclopentadien-l-yl) (Pentac®, dienochlor) tricyclohexyltin hydroxide (Plictran®, cyhexatin)

Nematicides 2-[diethoxyphosphinylimino]-l,3-diethietane

(fosthietan) S-methyl-1-(dimethylcarba oyl)-N-(methylcarbamoyl- oxy)-thioformimidate (Vydate®, oxamyl)

N-isopropylphosphoramidic acid, 0-ethyl-0'-[4-

(methylthio)-m-tolyl]diester (Nemacur®, fenamiphos)

Insecticides 3-hydroxy-N-methylcrotonamide(dimethylphosphate) ester (Azodrin®, monocrotophos) methylcarbamic acid, ester with 2,3-dihydro-2,2- dimethyl-7-benzofuranol (Furadan, carbofuran) 0-[2,4,5-trichloro-a-(chloromethyl)benzyl]phosphoric acid, 0' ,0'-dimethyl ester (Gardona, tetrachlor- vinphos) 2-mercaptosuccinic acid, diethyl ester, S-ester with thionophosphoric acid, dimethyl ester (malathion) phosphorothioic acid, 0,0-dimethyl, O-p-nitrophenyl ester (methyl parathion) methylcarbamie acid, ester with a-naphthol (Sevin®, carbaryl) methyl N-[ [ (methylamino)carbonyl]oxy]ethanimido- thioate (Lannate, methomyl) N'-(4-chloro-o-tolyl)-N,N-dimethylformamidine

(Fundal®, chlordimefor ) 0,0-diethyl-0-(2-isopropyl-4-methyl-6-pyrimidyl)- phosphorothioate (diazinon) octachlorocamphene (toxaphene)

O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN) cyano(3-phenoxyphenyl)-methyl 4-chloro- a-(1-methylethyl)benzeneacetate (Pydrin®, fenvalerate)

(3-phenoxyphenyl) ethyl (+)-cis,trans-3-(2,2-dichloro- ethenyl)-2,2-dimethylcyclopropanecarboxylate

(permethrin) dimethyl N,N'-[thiobis(N-methylimmo)carbonyloxy]]- bis[ethanimidothioate] (Larvin, thiodicarb) phosphorothiolothionic acid, 0-ethyl-0-[4-

(methylthio)phenyl]-S-n-propyl ester (Bolstar®, sulprofos) a-cyano-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylate (Fastac®, cypermethrin) cyano(3-phenoxyphenyl) ethyl 4-(difluoromethoxy)- α-(methylethyl) enzeneacetate (Pay-Off®, flucythrinate) 0,0-diethyl-0-(3,5,6-trichloro-2-pyridyl)phosphoro¬ thioate (Dursban®, chlorpyrifos) 0,0-dimethyl-S-[(4-oxo-l,2,3-benzotriazin-3-(4H)- yl)methyl]phosphorodithioate (Guthion®, azinphoε- methyl) 5,6-dimethyl-2-dimethylamino-4-ρyrimidinyl dimethyl carbamate (Pirimor®, pirimicarb) S-( -formyl-N-methylearbamoylmethyl)-0,0-dimethyl phosphorodithioate (Anthio®, formothion) S-2-(ethylthioethyl)-0,0-dimethyl phosphiorothioate (demeton-S-methyl) α-cyano-3-phenoxybenzyl cis-3-(2,2-dibromovinyl)- 2,2-dimethylcyclopropane carboxylate (Decis®, deltamethrin) cyano(3-phenoxyphenyl)methyl ester of N-(2-chloro- 4-trifluoromethylphenyl)alanine (Mavrik®, fluvalinate) .

The following Examples illustrate the invention.

EXAMPLES First Antibody Optimization for

Increased Sensitivity to Metsulfuron methyl The following definitions are used in the Examples:

"% CV" is the coefficient of variation, which is defined as:

Standard Deviation

CV - X 100.

Mean

"ma" refers to milli-absorbance units.

"Washing" refers to. rinsing at least two times with PBS/Tween-20® solution.

"PBS" refers to phosphate-buffered saline (see below) .

Tween-20® is a syrup of polyoxyethylene sorbitan esters available from the Sigma Chemical Company.

Tissuemizer® is a homogenizer manufactured by the TekMar Corporation.

O.D. refers to optical density. A. The coating conjugate coating step was conducted by dispensing 200 μl of coating conjugate at a concentration of 0.1 μg/ml in 0.1 M NaHC0₃, pH 9.4, in each well of a 96-well, flat-bottomed microwell plate and incubating at 4°C overnight. This time was chosen for convenience. Two hours at room temperature produces the same results. A concentration of 0.1 μg/ml coating conjugate was selected in an ELISA assay where a range of coating conjugate concentrations and a range of first antibody dilutions are assayed in various combinations (commonly referred to as a checker board ELISA). After optimal pair(s) are selected, i.e., an antibody dilution and a coating conjugate concentration that results in about 2 O.D.in about 1 hour. They are tested over the range of pesticide concentrations to be determined. The coating conjugate in the case of metsulfuron methyl was preferentially a metsulfuron methyl analog but may be 5-carboxy-metsulfuron methyl covalently coupled to ovalbumin. Other proteins can be used such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) depending on the hapten-carrier protein used as an immunogen.

The microwell plate was made of polystyrene. These plates are provided with a quality control certificate showing a physical and immuno-chemical control. Physical control: All wells were within +/-0.005 absorbance units from the mean. Immuno-chemical Control of homogenity is tested through adsorption of IgG. CV less than 5%. All results within +/- 10% from mean. B. Microplate Washing

A wash solution, consisting of phosphate- buffered saline (PBS) which contains 0.12 M NaCl, 2.7 mM KC1, 10 mM phosphate buffer at a pH of 7.5 at 25°C, and 0.05% Tween-20®, was used to remove excess coating conjugate from each well. Then, each well is filled with 200 μl of 3% BSA in PBS solution at room temperature and incubated for 2 hours to block the remaining sites. Excess blocking solution was then removed by washing and the plates are shaken to remove any droplets of moisture and stored at 4°C in sealed plastic bags.

C. First Antibody Reagent

The first antibody used in this assay was whole rabbit antiserum raised against an immunogen conjugate consisting of a carboxylic derivative of metsulfuron methyl, Compound (4), which was covalently coupled to the protein keyhole limpet hemocyanin (KLH) . The antiserum, diluted in PBS/0.5% BSA, comprises the first antibody reagent. Antibody dilutions of 1:100, 1:125, 1:165, 1:250 and 1:500 X 10³ were made and standard curves were generated with each of these antibody dilutions.

Corrpound (4) D. Labeled Second Antibody-Enzyme Conjugate

Alkaline Phosphatase conjugated Affinipure Goat Anti-Rabbit IgG (H&L) was prepared according to the manufacturer's instructions. The antibody concentration was 6 mg/ml and suggested dilution range is 1:5,000 to 1:50,000 for enzyme immunoassays using PNPP. In the standard ELISA assay, a 1:5,000 (.0012 mg/ml) dilution was made with PBS/0.5% BSA.

E. Enzyme Substrate Reagent The enzyme substrate was formed from 1 mg/ml p-nitrophenyl phosphate in diethanolamine buffer, pH 9.8. Diethanolamine buffer, 10%, consists of 97 ml of diethanolamine, 800 ml of water, 0.2 g of NaN , 100 mg of MgCl₂6H₂0; 1 M HC1 was added until the pH is 9.8. The total volume was made up to 1 liter with water and stored at room temperature in an amber bottle. The p-nitrophenyl phosphate is commercially available.

F. Assay Procedure

The assay was performed at room temperature and consisted of 1.0 ml of sample plus 0.8 ml PBS plus 0.2 ml of 10 X PBS/1% BSA for a total volume of 2.0 ml. The first antibody, 50 μl, was added to each sample. Standards were prepared similarly. The standards were 0, 0.005, 0.010, 0.025, 0.050 ng/ml. Each standard curve contained the antibody in one of the dilutions as described in C. After vortex-mixing and one hour preincubation, 200 μl of each reaction mixture was added to microplate wells in triplicate. After one hour incubation on the microplate, the plate was washed and 200 μl of second antibody-enzyme conjugate was added and incubated for 1 hour on the microplate. The microplate was washed again and 200 μl of substrate solution was added. A microplate reader was used to measure the absorbance at 405 n in each well of the microplate. These data were processed by a computer program which generated a standard curve based on. a four parameter logistic.

The negative control was yellow whereas the positive wells were very lightly-colored to colorless. To calculate % inhibition of the curve of each of the standards:

Negative Control Absorbance Standard Absorbance

100 = % Inhibition.

Negative Control Absorbance

where the negative control absorbance is the maximum O.D. measured in the absence of antigen and the standard absorbance is the O.D. obtained at the same time (as the negative control) with a known concentration of antigen. Thus, 0% inhibition indicates no analyte is present. When the percent inhibition exceeds about 15, accurate concentration can be easily determined.

G. Results

The following Table, generated as described above, shows the increased sensitivity of the assay employing improvement Step i of this invention. First Antibody Dilution X 10³

1:100 1:125 1:165 1:250 1:500

*<15%. At least 15% inhibition is necessary to obtain accurate measurements and distinguish from negative control.

Thus, it can be seen that high first antibody dilutions are useful for measuring low analyte concentrations. Additional studies showed that the antibody dilution of 1:500 X 10^ was optimal for a high sensitivity assay over a range of about 2.5 up to about 100 picrograms/ml. A further decrease in antibody concentration shows little improvement in sensitivity at higher pesticide concentrations but may improve sensitivity at lower concentrations.

First Antibody Optimization for Increased Sensitivity to Chlorimuron ethyl

Corrpound (8) A. Antigen Conjugate Coating and Blocking of Microplates

First, 200 μl of antigen-protein conjugate at a protein concentration of 0.1 μg/ml in 0.1 M NaHCθ3, pH 9.4 was placed in each well of a 96-well flat-bottomed microplate and incubated overnight at 4°C. The antigen. Compound (8), was a carboxylic acid derivative of an analog of chlorimuron ethyl, which was covalently coupled to the protein ovalbumin. A carboxylic acid derivative of chlorimuron ethyl itself can also be used. The washing, blocking and storage of the plates was the same as in metsulfuron methyl assay in Examples 1. The concentration of the coating conjugate was selected in a checkerboard assay as described in Example 1.

B. First Antibody Reagent

^HD2^C

Corrpound ⁵

The first antibody used in this assay was whole rabbit antiserum raised against an immunogen conjugate consisting of a carboxylic acid derivative of chlorimuron ethyl, Compound (5), which was covalently coupled to the protein keyhole limpet hemocyanin (KLH) . The antiserum diluted in PBS/0.5% BSA comprises the first antibody reagent.

C. Labeled Second Antibody-Enzyme Conjugate Reagent Affinity-purified goat anti-[rabbit IgG (H+L)]-alkaline phosphatase conjugate (Jackson Laboratories) was diluted 1:5000 (to 0.0012 mg/ml) with PBS/0.5% bovine serum albumin (BSA).

D. Enzyme Substrate Reagent

This was prepared as in the metsulfuron methyl assay of Example 1.

E. Assay Procedure The assay was performed substantially as in the metsulfuron methyl assay (Example 1) . Standards were prepared at 2.5, 5, 10, and 50 picogram/ml of chlorimuron ethyl by adding 20 μl of an appropriate concentration stock solution to 1.8 ml of water only. To each standard was added 200 μl of 10% PBS/1% BSA and 10 μl of one of the four first antibody reagent dilutions, then each tube was vortex-mixed and incubated at room temperature for one hour.

F. Results

The following data demonstrates the increased sensitivity of the assay resulting from the decreased antibody concentration. Final Antibody Dilution X lO³-

64 128 320 640

These results demonstrate the improved sensitivity obtained by diluting first antibody. Optimal sensitivity at low concentrations and optimal dynamic range were obtained using the 1:320,000 final antibody titer. Further reduction in antibody concentration did not improve sensitivity at low concentrations, and reduced the overall dynamic range of the assay.

Optimization of Second Antibody-Alkaline Phosphatase Conjugate Concentration

A. The ELISA procedure was exactly as described above with the exception that first antibody dilution was 1:500 X 10^ in the assay in all cases; and second antibody-enzyme conjugate concentration was 1.2, 2.4 or 4.8 μg/ml. The concentration of 1.2 μg/ml is an art-recognized concentration whereas those of 2.4 and 4.8 are about 2X and 4X more concentrated than the art would suggest.

The following data show little change in the percent inhibition for each of the standards with the three concentrations of second antibody-enzyme conjugate but a significant decrease in time for the assay to develop was seen with increased concentration.

Further increases in second antibody-enzyme conjugate did not result in any further decreases in substrate incubation time.

Optimization of Assay Time bv Increasing Second Antibody Concentration

A. Reagents and Procedure

The antigen conjugate coating and blocking of microplates was carried out as described above. The first antibody reagent used was diluted by the optimal dilution factor determined above, to a final titer of 1:320,000 in the assay. The labeled antibody-enzyme conjugate reagent was prepared as above and diluted so that 1.2, 2.4 and 4.8 μg/ml conjugate concentrations were used in the assays. B . Results

The following data show the decreased substrate incubation time resulting from the increased concentration of labeled antibody-enzyme conjugate.

Conjugate Substrate Incubation Time Micrograms/ml minutes Control 1.2 156 Ex. 3 2.4 124

Ex. 4 4.8 102

The optimal conjugate concentration was 4.8 micrograms/ml. The assay sensitivity and dynamic range were not affected by changes in the conjugate concentration.

EXAMPLES 5 AND 6

A. Precision

The ELISA procedure was as described with the first antibody dilution at 1:100 X 10³ and 1:500 X 10³ for titer 1:100,000 and 1:500,000, the standard and high sensitivity assay, respectively. The labeled second antibody enzyme conjugate concentration was at 1.2 μg/ml for the standard assay and at 4.8 μg/ml for the high sensitivity assay.

This result clearly demonstrates that the precision of the assay, as indicated by % CV, actually improves at lower concentrations in the high-sensitivity format described in this invention. Under these conditions, at higher sulfonylurea concentrations of 25, 50 and 100 pg/ml there was an indication of greater precision with the standard assay than with the assay of this invention.

Employing the ELISA procedure of this invention, the presence of metsulfuron methyl was detected and measured at about 100 pg/ml in the following media: apple juice, apricot juice, white grape juice, orange juice, tomato juice, nectarine fruit, and tomato fruit.

Likewise, metsulfuron methyl was measured at between 10 to 50 pg/ml in soil of the following types (after extraction into an aqueous medium): TAMA, pH - 6.5 to 7.0, organic matter = 2.6%; SASSAFRAS, pH = 6.0, organic matter = 0.1%; and FARGO, pH = 7.6, organic matter = 6%.

Likewise, metsulfuron methyl was measured at about 100 pg/ml (after extraction into an aqueous medium) in corn forage and corn stover.

Claims

CLAIMSWhat is claimed is:

1. An improved antigen-capture enzyme-linked immunosorbent assay, for measuring the presence of a target pesticide in a medium, comprising the steps of (a) forming a complex of the pesticide in the medium with an excess of a first antibody of known titer;

(d) determining the amount of pesticide in the medium by comparing the signal generated by the labeled antibody of Step (c) to the signal generated by running the above steps with a sample containing no pesticide and one or more samples containing known concentrations of pesticide; wherein the improvement comprises: (i) diluting the titer of the first antibody employed in Step (a) such that the concentration of pesticide needed to reduce the signal generated by the label in Step (d) by 50% is less than one-half the concentration required without the dilution; and (ϋ) increasing the titer of the labeled antibody employed in Step (c) so that the total time necessary to run the assay is about the same as a normal assay without the dilution of (i); thereby

(iii) increasing by a factor of at least two the sensitivity of the assay to detect target pesticide.

2. An assay according to Claim 1 wherein the pesticide is a sulfonylurea.

3. An assay according to Claim 2 employing a coating conjugate in Step (b) comprising a carboxylated sulfonylurea or derivative thereof covalently linked via the carboxyl group to a protein,

4. An assay according to Claim 3 wherein the protein is selected from the group keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, pumpkin seed globulin or marijuana seed globulin.

5. An assay according to Claim 4 wherein the sulfonylurea is selected from the group of Compounds (1) to (14).

6. An assay according to Claim 1 wherein the medium is water.

7. An assay according to Claim 1 wherein the medium is soil.

8. An assay according to Claim 1 wherein the medium is a crop matrix.

9. An assay according to Claim 1 wherein the medium is a food matrix.

10. An assay according to Claim 1 wherein the concentration of the target pesticide is no more than about 100 picograms per ml.

11. An assay according to Claim 10 wherein the concentration of the target pesticide is no more than about 10 picograms per ml.

12. An assay according to Claim 1 wherein the first antibody is a rabbit polyclonal antibody raised against a carboxylated sulfonylurea or derivative thereof covalently linked via the carboxyl group to a protein selected from the group keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, pumpkin seed globulin, or marijuana seed globulin.

13. A kit useful for detecting and measuring a target pesticide in an unknown sample comprising the components:

(i) an antibody to the target pesticide in the unknown; (ii) a solid phase having a coating conjugate bound to it;

(iii) a labeled antibody that recognizes antibody (i);

(iv) a developer that develops color in the presence of a label; and

(v) controls comprising at least one known concentration of the pesticide and one containing no pesticide, the components cooperating so that upon contacting i, ii and iii with iv a color is developed which indicates, upon comparison to the controls, the presence and concentration of the target pesticide; wherein the titer of the antibody in component (i) is such that the concentration of the pesticide needed to reduce the signal generated by the label by 50% is less than half the concentration required without the dilution, and the titer of the labeled antibody in component (iii) is such that the time necessary to measure the pesticide is about the same as in a normal assay without the dilution of antibody in component (i) .

14. A kit according to Claim 13 wherein the target pesticide is a sulfonylurea.

15. A test kit according to Claim 14 comprising an antibody to the target sulfonylurea selected from the group chlorsulfuron, bensulfuron, metsulfuron methyl and chlorimuron ethyl.

16. A kit according to Claim 13 wherein the target pesticide is benomyl.

17. A method for using the kit according to Claim 13 to detect a target pesticide comprising contacting components i, ii, iii and iv and comparing the color that is developed to controls, v, thereby determining the presence and concentration of the target pesticide.