CA2067342A1 - Method for detecting pesticides at the picogram level - Google Patents

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

W~91/05259 2 0 S 7 3 ~ 2 PCT/US90/05424 TITLE
IMPROvED METHOD FOR DETECTING
PESTlCIDES AT THE PICOG~M LEVEL

~ack~round of th~ Inve!ntion A variety of enzyme-linked i~nunosorbent assay (ELISA) formats have been employed to detect pesticide~. 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 reductîon 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: ~elley et al., ~l. A~ric. FQod Chem., 19~5, 33, pages 962 to g65.
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, 1983.

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WO91/OS259 %~ ~7 ~ ~ PCT/~S90/05424 _ SummarY of the Invention This invention concerns an improved S 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 comple~ of the pesticide in the medium with an e~cess of a first antibody of known titer;
~ b) binding the free antihody 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 con~ugate 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.

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WO91105~59 2 ~ S 7 3 ~ 2 PCT/US90/~5424 ~, . . .

~ y ~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 t~e practical application of this technique to obtain high sensitivity. However, an interrelated aspect of the present invention provides for an increase in concentration o~ 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 l to lO0 picogram/ml range in an aqueous medium. Samples containing higher concentrations may be diluted into this range, which offers the adYantage of significant reduction in errors introduced by too-high concentrations of unknown materials in the medium; that is, a reduction in matri~ 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 .

' WO91/05259 ~ P~T/US90/05424_ 2 ~ 7~ 2 4 polyclonal antibody produced by methods described hereafter. Preferred labeled antibody-enzyme conjugate is alkaline phosphatase-goat anti-rabbit Ig~. Preferred protein-antigen conjugates (coating conjugates) are described hereafter. Preferred as a solid phase support is a 96-well microtiter plate.

~etails of ~he 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 }5 containing the pesticide, whereby a fraction of said antibody forms a comple~ with the pesticide and the balance remains free. The free antibody is then separated from the reaction mi~ture 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. Aaric. Food Ch~m., 1982, ~0, pages 572 to 580 studied different hapten structures for parathion. He concluded that "the determinant groups of the small molecule must be preserved~ and , ,.
: ' . ' ' ;,'''. . ~ .' ' ' . :' : . .` ' WO~1/05259 PCT/VS90/~5424 2 067 3 ~2 that ~the hapten~s determinant groups must not be masked~' for antibody production. The format most commonly used for pesticide i~unoassays has utilized a solid phase with hapten bound to it (coating conjugate) for capture of antibodies not bound to free compound. The quantification o]E the captured antibody is used to determine the original concentration of compound in the original aqueous sample.
Hapten structures were further e~plored by Wie et 21 ., J. Aqric. Food ~hem-, 1984, ~, pages 1299 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 thar. the immunizing hapten. In particular, they demonstrated a different linker arm and different position of linker arm gave Z0 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. ~VII, 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 . ~ ., : . ,. . ~ . .

wo9l/os2ss PCT/US90/05424 _ ; 20~3~2 6 for immunization is important, particularly preserving the common antigenic determinants of the S related compounds.
In the instant invention, the hapten in the coatin~ 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 carbo~ylate moiety is used for attachment of the hapten to the coating protein. The best coating protein/hapten combination i5 determined empirically based on maximizing affinity for the first antibody.
E~amples of haptens that are useful in this invention are as follows:

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~N~

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~ ~NH ~ C~`cco~ ~Z~

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OOC~19 ~ HOCC~[~S`NHJ~NH~OCH3 CC)OCH~

HOOC~W~ 5) COOC~12CH, .. . .
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~Q6-73~2 8 . ., OCH( CH3~)2' 10~ ~ `NH ~~ ~ ~l OCH~C~13~z ( HOOC

7) H~C
-Cl o~\~o,Ll~n3 t0~ ~:

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2 0 ~H~SO, NHCMH~(N ( 1 3 ) 02cH3 OCH2CO2H

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~C~CH3 CO,~ , 11 20`~73~2 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 St:ep (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 o~ides, polymeric materials such as agarose, polystyrene, polyacrylamide, their derivatives or mixtures ; 20 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 mi~ture and the treated solid phase material become separated into a solid phase containing the coating conjugate - bound with any e~cess first antibody which will ' , .
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' W091/~5259 PCT/~S9~/05424 -- :`20S73~2 12 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., Ana~Ytical - ~ioch~m, 1~1, 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 z5 "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 i5 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 introducin~
a labeled second antibody which is directed against the Fc portion of the bound first antibody. Typical labels include enzymes, radioisotopes, chromophores, ::
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13 2~73~2 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 comple~es are known in the art of diagnostic immunoassay and are generally discussed in Miles et al., "Labelled Antibodies and Immunological Assay Systems", Na~ure, , pag~s 187 to 189 (1968) and U.';. 3,654,090. The preferred label in the instant invention is an anti-rabbit antibody-alkaline phosphatase conjugate and a p-nitrophenyl phosphate substrate or 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 o this invention include:

2-chloro-N-~(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl}benzenesulfonamide Chlorsuluron methyl2-[[[[~4,6-dimethyl-2-pyrimidinyl~amino]-carbonyl]amino]sulfonyl]benzoate Sulfometuron methyl methyl-2-~[~[(9-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate Metsulfuron methyl 2-[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylamino]carbonyl]amino]sulfonyl]benzoic acid, methyl ester ; E~press (TM) ethyl 2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl), amino]carbonyl~amino]sulfonyl]benzoate Chlorimuron ethyl ~. . . ..
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WO91/05259 PCT/U~90/0~424_ ~20&~3~ 14 2-~(4-etho~y-6-methylamino-1,3,5-triazin-2-yl)aminocarbonyl]aminosulfonyl~benzoic acid, S methyl ester Muster (TM) 2-[[(4,6-dimethoxy-1,3,5-triazin-2-yl)aminocarbonyl]-aminosulfonyl]-4-(2,2,2-trifluoroetAoxy)benzoic acid, ethyl ester 10 4-chloro-2-[~(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]aminosulfonyl]benzoic acid, isopropyl ester 3-[~[[(4-metho~y-6-methyl-1,3,5 tria~in-2-yl)amino]-carbonyl~amino]sulfonyl]-2-thiophene carboxylic acid, methyl ester ..
Thiameturon methyl methyl 2-[[[[(9,6-dimethoxy-2-pyrimidinyl)amino}-carbonyl]amino]sulfonyl]methylbenzoate Bensulfuron methyl 2-[~(4~6-dimethoxypyrimidin-2-yl)aminocarbonyl]-aminosulfonyl]N,N-dimethyl-3-pyridinecarbo~amide 2-[[(4,6-dimetho~ypyrimidin-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 :30 2-~[[(4,6-bis(difluoromethoxy)-2-pyrimidinyl]-amino]carbonyl]amino]sulfonyl]benzoic acid, methyl ester ethyl S [3-(~,6-dimethoxypyrimidin-2-yl)ureido-sulfonyl~-l-methylpyrazole-4-carboxylate N-[(6-methoxy-4-methyl-1,3,5-triazin-Z-yl)amino-carbonyl]-2-(2-chloroethoxy~benzene sulfonamide : . . . - . .

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- . , WO91/05259 PCT/US90/054~4 ``. ; ~2~673~2 N-[(4,6-dimethoxy-1,3,5-triazin-2-yl)amino-carbonyl]-2-(2-methoxyetho~y)benzenesulfonamide N-[(4,6-dimethoxypyrimidin-2-yl)-amino]carbonyl]-3-trifluoromethyl-2-pyridinesulfonamide.

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

Common Name Chemical Name ace~ochlor 2-chloro-N-(ethoxymethyl)-N(2-ethyl-6-methylphenyl)-acetamide acifluorfen 5-[2-chloro-4-(trifluoromethyl)-phenoxy]-2-nitrobenzoic acid acrolein 2 propenal 20 alachlor 2-chloro-N-(2,6-diethylphenyl)-N(methox~methyl)acetamide ametryn N-ethyl-N'-(l-methylethyl)-6-~methylthio)-1,3,S triazine-2,4diamine 25 amitrole lH-1,2,4-triazol-3-amine AMS ammonium sulfamate asulam methyl [(4-aminophenyl)sulfonyl]-carbamate atrazine 6-chloro-N-ethyl-N'-(l-methyl-ethyl)l,3,5-triazine-2,4-diamine barban 4-chloro-2-butynyl 3-chloro-carbamate benefin N-butyl-N~ethyl-Z,6-dinitro-4-(trifluoromethyl)benzenamine bensulide O,O-bis(l-methylethyl) S-[2[(phenylsulfonyl~amino]~
ethyl]phosphorodithioate , .~
.

WO91/05259 PCT/US90/05424 ~
2~7342 15 bentazon 3-~1-methylethyl)-(lH)-2,1,3-benzothiadiazin-4(3H)-one, 2,2-dioxide benzofluor N-[4-(ethylthio)-2-(trifluoro-methyl)phenyl]methane-sulfonamide benzoylprop N-benzoyl-N-(3,4-dichlorophenyl)-DLalanine -bifeno~ methyl 5-(2,4-dichloro-phenoxy)-2nitrobenzoate bromacil 5-bromo-6-methyl 3-(1-methylpro-pyl)2,4(1H,3H)pyrimidinedione 15 br~moxynil 3,5-dibromo-4-hydroxybenzonitrile butachlor N-(buto~ymethyl)-2-chloro-N-(2,6diethylphenyl)acetamide buthidazole 3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2imidazolidinone butralin 4-(1,1-dimethylethyl)-N-(l-methylpropyl)-2,6-dinitro-benzenamine butylate S ethyl bis(2-methylpropyl)car-bamothioate cacodylic dimethyl arsinic oxide acid CDAA 2-chloro-N,N-di-2-propenyl-acetamide ; CDEC 2-chloroallyl diethyldi-thiocarbamate chloramben 3-amino-2,5-dichlorobenzoic acid chlorbromuron 3-(9-bromo-3-chlorophenyl)-1-. metho~y-lmethylurea chlorimuron 2-[[[[(9 chloro-6-methoxy-2-~` 35 ethyl pyrimiethyldinyl)ethylamino]-carbonyl]amino]sulfonyl]benzoic ~ acid, ethyl ester , .
"~ :- ...

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17 2~;6~;3~2 chloro~uron N~-[4-(4-chlorophenoxy)phenyl]-N,Ndimethylurea 5 chlorpropham l-methylethyl 3-chlorophenyl-carbamate chlortoluron N'-~3-chloro-4-methylphenyl)-N,Ndimethylurea cinmethylin exo-l-methyl-4-(1-methylethyl)-2-[(2methylphenyl)methoxy]~7-o~abicyclo[2.2.1]heptane clethodim (E,E)-(+)-2-[1-~[~3~chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one clomazone 2-~(2-chlorophenyl)methyl]-4,4-dimethyl3-iso~azolidinone clopro~ydim (E,E)-2-[1-[[(3-chloro-2-pro-penyl)o~y)imino]butyl]-5-[2-(ethylthio)propyl]3-hydroxy-2-cyclohexen-1-one clopyralid 3,6-dichloro-2-pyridinecar-bo~ylic acid CMA calcium salt of MAA
25 cyanazine 2-[[4-chloro-6-(ethylamino)-1,3,5-triazin-2-yl~amino]-2-methylpropanenitrile cycloate S-ethyl cyclohe~ylethylcar-bamothioate 30 cycluron 3-cyclooctyl-1,1-dimethylurea cyperquat l-methyl-4-phenylpyridinium cyprazine 2-chloro-4-(cyclopropylamino)-6-(isopropylamino)-s-triazine cyprazole N-[5-(2-chloro-1,1-dimethyl-ethyl)-1,3,4thiadiazol-2-yl]cyclopropanecarboxamide .

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. ' : . ' ;'' ,: , ' ', WO91/052~9 PCT/US90/05424 _ 2067'~2 18 cyprcmid 3~,4~-dichlorocyclopropane-carbo~anilide 5 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-clicarbo~ylate 10 desmediphan ethyl [3-[[(phenylamino)-carbonyl]oxyJphenyl]carbamate desmetryn 2-~isopropylamino)-4-(methyl-amino)-6(methylthio)-s-triazine 15 diallate 5-(2,3-dichloro-2-propenyl)-bis(lmethylethyl)carbamothioate dicamba 3,6-dichloro-2-~etho~ybenzoic acid dichlobenil 2,6-dichlorobenzonitrile 20 dichlorprop (+)-2-(2,4-dichloropheno:~y)-propanoic acid dichlofop (+)-2-[4-(2,4-dichlorophenoxy)-phenoxy]propanoic acid diethatyl N-(chloroacetyl)-N-(2,6-diethyl-phenyl)glycine difenzoquat 1,2-dimethyl-3,5-diphenyl-lH-pyrazolium dinitramine N3,N3-diethyl-2,4-dinitro-6-(trifluoromethyl)-1,3 benzenediamine dinoseb 2-(1-methylpropyl)-4,6-dinitro-phenol diphenamid N,N-dimethyl-a-phenylbenzene-acetamide '. ' : .' - . ::. ~ ~ ,,:' .. . . . . .. . . .

WO~1/05259 PCT/US9~/05424 19 20~73~

dipropetryn 6-(ethylthio)-M,N'-bis~l-methyl-ethyl)l,3,5-triazine-2,4-diamine diquat 6,7-dihydrodipyrido[1,2-a:2',1'-- c]pyrazinedium ion diuron N'-(3,4-dichlorophenyl)-N,N-dimethylurea 10 DNOC 2-methyl-4,6-dinitrophenol DSMA disodium salt of MAA
endothall 7-o~abicyclo[2.2.1]heptane-2,3-dicarbo~ylic acid EPTC S-ethyl dipropylcarbamothioate 15 ethalfluralin N-ethyl-N-(2-methyl-2-propenyl) : 2,6dinitro-4-(trifluoro- -methyl)benzenamine ethofumesate (+)-2-ethoxy-2,3-dihydro-3 t 3-dimethyl5-benzofuranyl methanesulfonate fenac 2,3,6-trichlorobenzeneacetic acid fenoxaprop (+)-2-[4-[(6-chloro-2-benzoxa-zolyl)o~y]pheno~y]propanoic acid 25 fenuron N,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-; 30 pyridinyl]o~y]phenoxy]pro-panoic acid fluazifop-P (R)-2-[4 [[5-(trifluoromethyl)-2-pyridinyl]oxy~pheno~y]pro-panoic acid ; 35 ., . . -. . . . . . .

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W09l/05259 PCT/US90/05424 ~673~2 20 .
fluchloralin N-(2-chloroethyl)-2,6-dinitro-N-propyl4-(trifluoromethyl)-benzenamine fluometuron N,N-dimethyl-N'-[3-(trifluoro-methyl)phenyl]urea fluorochlor~ 3-chloro-4-(chloromethyl)-1-[3-idone (trifluoromethyl)phenyl]-2-pyrrolidinone fluorodifen p-nitrophenyl a, a, -trifluoro-2-nitro-p-tolyl ether fluorogly- . carbo~ymethyl 5-[2-chloro-4-cofen (trifluoromethyl)phenoxy]-Z-nitrobenzoate fluridone l-methyl-3-phenyl-5-[3-(tri-fluoromethyl)phenyl]-4(1H)-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 halo~yfop 2-[4-[[3-chloro-5-(trifluoro-methyl)-2-pyridinyl~oxy]-phenoxy]propanoic acid he~aflurate potassium hexafluoroarsenate hexazinone 3-cyclohexyl-6-(dimethylamino)-l-methyll,3,5-triazine-2,4(1H,3H)-dione - - , . , . . . . . ~ . : . .

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WO91/0525~ PCT/US90/05424 21 ` 2~342 imazametha- 6-(~-isopropyl-4-methyl-5-oxo-benz 2-imidazolin-2-yl)-m-toluic acid, methyl ester and 6-{4-isopropylq-methyl-5-o~o-2-imidazolin-2-yl)p-toluic acid, methyl ester imazapyr (~-2-[~,5-dihydro-4-methyl-4-(1-methylethyl)-.5-o~o-lH-imidazo1-2-yl]-3pyridine-carboxylic acid imazaquin . 2-[4,5-dihydro-4-methyl-4-~l-methylethyl)-5-o~o-lH-imidazol-2-yl]-3quinoline-carboxylic acid imazethapyr (+)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-lH-imidazol-2-yl]-5ethyl-3-pyridinecar-bo~ylic acid iogynil 4-hydro~y-3,5-diiodobenzonitrile isopropalin 4-(1-methylethyl)-2,6-dinitro-N,Ndipropylbenzenamine isoproturon N-(~-isopropylphenyl)-N~
dimethylurea isouron N'-[5-(1,1-dimethylethyl)-3-isoxazolylJN,N-dimethylurea iso~aben N-[3-(1-ethyl-1-methylpropyl)-5isoxazolyl]-2,6-dimethoxy-benzamide karbutilate 3-[~(dimethylamino~carbonyl]-amino]phenyl-(l,l-dimethyl-ethyl)carbamate lactofen (~)-2-etho~y-1-methyl-2-o~o-ethyl 5-[2chloro-4-(trifluoro-methyl)phenoxy]2-nitrobenzoate .

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WO9~/052~9 PCT/US90/OS424_~
20~ 3 42 22 lenacil 3-cyclohexyl-6,7-dihydro-lH-cyclopentapyrimidine-2,4(3H,5H)-dione linuron N'-(3,4-dichlorophenyl)-N-methoxy-Nmethylurea MAA methylarsonic acid MAMA monoammonium salt of MAA
10 MCPA (4-chloro-2-methylpheno~y)acetic acid MCPB 4-(4-chloro-2-methylphenoxy)-- butanoic acid mecoprop (+)-2-(4-chloro-2-methylphenogy)-propanoic acid mefluidide N-[2,4-dimethyl-5-[C(trifluoro-methyl)sulfonyl]amino]phenyl]-acetamide methal- N-(2-methyl-2-propenyl) 2,6-propalin dinitro-N-4-(tri~
fluoromethyl)benzenamide methabenz- 1,3-dimethyl~3-~2-benzothia-thiazuron zolyl)urea metham methylcarbamodithioic acid 2S methazole 2-(3,4-dichlorophenyl)-~-methyl-1,2,40xadiazolidine-3,5-dione methoxuron N'-(3-chloro-4-methoxyphenyl)-N,Ndimethylurea metolachlor 2-chloro-N-(2-ethyl-6-methyl-phenyl)-N(2~methoxy-1-methylethyl)acetamide metribuzin 4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one 35 MH 1,2-dihydro-3,6-pyridazinedione - . .
' . ' ~' ' .~, ~ ' .', , ,, ' , . . .

:. , : ' :
' W~91/Q52~9 PC~/~S90/05424 molinate S-ethyl he~ahydro-lH-azepine~
l-carbothioate 5 monolinuron 3-(p-~hloropheny1)-1-methoxy-1-methylurea monuron N'-(4-chlorophenyl)-N,N-dimethyl-urea monuron TCA Salt of monuron and TCA
10 MSMA monosodium salt of MAA
napropamide N,N-diethyl-2-(1-naphthalenyl-o~y)propanamicle naptalam 2-[(1-naphthalenylamino)carbo-nyl~benzoic acid 15 neburon 1-butyl-3-(3,9-dichlorophenyl)-l-methylurea ~itralin 4-(methylsulfonyl)-2,6-dinitro-N,Ndipropylaniline nitrofen 2,4-dichloro-1-(g-nitrophenoxy)-benzene nitrofluorfen 2-chloro-1-(4-nitropheno~y~-4-(trifluoromethyl)benzene norea N,N-dimethyl-N'-(octahydro-4,7-methanolH-inden-5-yl)urea 3a~, 4a, 5a, 7a, 7aa-isomer norflurazon 4-chloro-5-(methylamino)-2-~3-(trifluoromethyl)phenyl]-3t2H)pyridazinone oryzalin 4-tdipropylamino)-3,5-dinitro-benzenesulfonamide o~adiazon 3-[2,4-dichloro-S-(l-methyl-ethoxy)phenyl]-5 ( 1, 1-dimethylethyl)l,3,4-o~a-diazol-2(3H)-one :' ~ '' . ' ~ ,' . -. ', . ' , . ': ~ :
' ' : ' ', ~

WO91/052~9 PCT/US90/05424 .: ~
20~73~2 ~4 oxyfluorfen 2-chloro-1-(3-ethoxy-4-nitro-pheno~y)-4-(trifluoromethyl)- :
benzene paraquat l,l'-dimethyl-4,4'-dipyridin-ium ion pebulate S-propyl butylethylcarbamothioate pendimethalin N-(l ethylpropyl)-3,4-dimethyl-2,6dinitrobenzenamine per1uidone 1,1,1-trifluoro-N-Z2-methyl-4-(phenylsulfonyl)phenyl]methane-sulfonamide phenmedipham 3-[(metho~ycarbonyl)amino]phenyl-(3methylphenyl)carbamate picloram 4-amino-3,5,6-trichloro-2-pyridinecarbo~ylic acid PPG-1013 5-[2-chloro-4-(trifluoromethyl)-pheno~y]-2-nitroacetophenone o~ime-0-acetic acid, methyl ester procyazine 2-[[4-chloro-6-(cyclopropyl-~ amino)-1,3,5triazine-Z-; yl]amino]-2-methylpropane : 2S nitrile profluralin N-(cyclopropylmethyl)-2,6-dinitro-Npropyl-4-(tri-fluoromethyl)benzenamine prometon 6-methoxy-N,N'-bis(l-methyl-ethyl)-1,3,5triazine-2,4-diamine prometryn N,N'-bis(l-methylethyl)-6-(methylthio)1,3,5-triazine-2,4- :
diamine 35 pronamide 3,S-dichloro-N-(l,l-dimethyl-2-~ propynyl)benzamide .

, - , . . . .
; : "

: " ' ' , ~ . .
. :. .

WO~1/0~259 PCr/US90/05424 propachlor 2 chloro-N-(l-methylethyl)-N-phenylacetamide 5 propanil N-(3,4-dichlorophenyl)propanamide propazine 6-chloro-N,N'-bis(l-methylethyl)-1,3,5-triazine-2,4-diamine propham l-methylethyl phenylcarbamate prosulfalin N-[[4-(dipropylamino)-3,5-dinitrophenyl]sulfonyl]-S,S-dimethylsulilimine prynachlor 2-chloro-N-(l-methyl-2-pro-. pynyl)acetanilide pyrazon 5-amino-4-chloro-2-phenyl-3(2H)pyridazinone quizalofop (+)-2-[4-[(6-chloro-2~
quino2alinyl)ethylo~y]-pheno~y]propanoic acid, ethyl ester 20 secbumeton N-ethyl-6-metho~y-N'-(l-methylpropyl)l,3,5-triazine-2,4-diamine setho~ydim 2-[1-(etho~yimino)butyl]-5- :
[2-(ethylthio)propyl]-3-hydroxy-2-cyclohe~en-1 one siduron N-~2-methylcyclohexyl)-N~-phenylurea simazine 6-chloro-N,N'-diethyl-1,3,5-triazine2,4-diamine 30 sulfometuron 2-[[[[(4,6-dimethyl-2-pyrimi-dinyl)methylamino]carbonyl]-amino]sulfonyl]benzoic acid, methyl ester TCA trichloroacetic acid . ' ' ' ' ' ' , '' :
.

.

W~91/~52~9 PCT/U~0/05424 ~
2~73`~2. 26 tebuthiuron N-[5-(1,1 dimethylethyl) 1,3,4-thiadiazol-2-yl]-N,M'-dimethylurea terbacil 5-chloro-3-(1,1-dimethylethyl3-6-methyl-2,4(1H,3H)-pyrimidi~edione terbuchlor N (buto~ymethyl)-2-chloro-N-[2-.(l,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-(l,l-dimethylethyl)-N'-ethyl-; 6(methylthio)-1,3,5-triazine-2,4-diamine thiobencarb S-[(4-chlorophenyl)methyl]
diethylcarbamothioate triallate 5-(2,3,3-trichloro-2-propenyl)-bis(lmethylethyl)carbamothioate triclopyr [(3,5,6-trichloro-2-pyridinyl)-I oxy]aeetic acid : 25 tridiphane 2-(3,5-dichlorophenyl)-2-(2,2,2trichloroethyl)oxirane trifluralin 2,6-dinitro-N,~-dipropyl-4-(trifluoromethyl)benzenamine trimeturon l-(p-chlorophenyl)-2,3,3-tri-', 30 methylpseudourea 2,4-D (2,4-dichlorophenoxy)acetic acid 2,4-DB 4-(2,4-dichlorophenoxy)butanoic ~ acid vernolate S-propyl dipropylcarbamothioate 35 ~ylachlor 2 chloro-N-(2,3-dimethylphenyl)- A
; N(l-methylethyl)acetamide ,: .: : -: . . .
; '~
~, .

~091t05259 PCT/US90/05424 27 2~6734~

Funqicides methyl 2-benzimidazolecarbamate (carbendazim) tetramethylthiuram disulfide (thiuram) n-dodecylguanidine acetate (dodine) manganese ethylenebisdithiocarbamate (maneb) 1,4-dichloro-2,5-dimethoxybenzene (c:hloroneb) methyl l-(butylcarbamoyl)-2-benzimiclazolecarbamate (benomyl) 2-cyano-N-ethylcarbamoyl-2-metho~yiminoacetamide (cymo~anil) N-trichloromethylthiotetrahydrophthalamide (captan) N-trichloromethylthiophthalimide (folpet) dimethyl 4,4'-(o-phenylene)bis(3-thioallophanate) (thiophanate-methyl~
2-(thiazol-4-yl)benzimidazole (thiabendazole) aluminum tris~O-ethyl phosphonate) (phosethyl aluminum) tetrachloroisophthalonitrile (chlorothalonil) 2,6 dichloro-4 nitroaniline (dichloran) N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine methyl ester (metalaxyl) cis-N-[1,1,2,2-tetrachloroethyl)thio}cyclohe~-4-ene-1,2-dicarbio~imide (captafol) 3-(3,5-dichlorophenyl)-N-(l-methylethyl)-2,4-dio~o-l-imidazolidine carbo~amide (iprodione) 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl-2,4-o~azolidinedione (vinclozolin) kasugamycin O-ethyl-S,S-diphenylphosphorodithioate (edifenphos) 4-(3-(4-(1,1-dimethyl-ethyl)phenyl)-2-methyl)propyl-2,6-dimethylmorpholine (fenpropimorph) 4-(3-4(1,1-dimethyl-ethyl)phenyl)-2-methyl)propylpi peridine (fenpropidine) . .
.

WO91/0~59 PCT/US9OlO~24 20~34Z 28 1-(4-chlorophenoxy)-3,3-dimethyl-1-(lH-1,2,9-triazol-l-yl)butanone (Bayleton, triadimefon) R-(4-chlorophenoxy)-a-(1,1-dimethylethyl) 1-H-1,2,4-triazol-l-ethanol (Baytan, triadimenol) 2-(4-chlorophenyl)-2-(lH-1,2,4-triaz:ol-1-ylmethyl)-hexanenitrile (Systhane0, myclobutanil) Folicur~
(tebuconazol) 3-chloro-4-[4-methyl-2-(lH-1,2,4-triazol)-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl-4-chlorophenyl ether (Score~) 1-~2-(2,4-dichlorophenyl)pentyl]lH-1,2,4-triazole (Topas~, penconazole) +-a-(2-fluorophenyl-a-(4-fluorophenyl)-lH-1,2,4-triazole-1-ethanol (Impact~, flutriafol) l-[[bis(4-fluorophenyl)methylsilyl)methyl]-lH-1,2,4-triazole (Nustar~, flusilazol) l-N-propyl-N-[2(2,4,6-trichlorophenoxy)ethy:l]-carbamoylimidazole (Sportak~, prochloraz) 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl~methyl]-lH-1,2,4-triazole (Tilt~, propiconazole) (+/-)-a-butyl-a-(2,4-dichlorophenyl)-lH-1,2,4-triazole-l-ethanol ~Anvil~, hexaconazole) ~-(2-chlorophenyl)-a-(4-chlorophenyl)-5-pyridine-methanol (Rubigan~, fenarimol) methyl N-(2,6-dimethylphenyl)-N (2-furanylcarbonyl)-DL-alaninate (Fongarid~, furalaxyl) Bact~ric,ldes streptomycin sulfate oxytetracycline (dihydrate) ~5 : ~, . ......

`, '.

W~91/05259 ~ PCT/US90/05424 Acaricides trans-5-(4-chlorophenyl)-N-cyclohexyl-4-methyl-2-o~o-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~, oxythioquino~) 2,2,2-trichloro-l,l-bis(4-chlorophenyl)ethanol (Kelthane~, dicofol) bis(pentachloro-2,4-cyclopentadien-l-yl) (Pentac~, dienochlor) tricyclohe~yltin hydroxide (Plictran~, cyhexatin) Nemati~ides ~2-[dietho~yphosphinylimino]-l,3-diethietane ; (fosthietan) S-methyl-l-(dimethylcarbamoyl) N-(methylcarbamoyl-oxy)-thioformimidate (Vydate~, oxamyl) N-isopropylphosphoramidic acid, 0-ethyl-0'-[4-(methylthio)-m-tolyl]diester (Nemacur~, fenamiphos) Inseç~ici~es 3-hydrosy-N-methylcrotonamide(dimethylphosphate) ester (Azodrin~, monocrotophos) methylcarbamic acid, ester with 2,3-dihydro-2,2-dimethyl-7-benzofuranol (Furadan, carbofuran) 0-[2,~,5-trichloro-a-(chloromethyl)benzyl]phosphoric 30acid, 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, 0-p-nitrophenyl ; 35ester (methyl parathion) :' ' .' :
' .. , . , ~ -' .: ' ' ' ' ' ' ." . . , ' ' ~ ....... ' : ~

- ., . ' ~ ' ' ' ' . , W091/052~9 PCT/U~90/05~24 _ 2~6`73~ 30 methylcarbamic 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~, chlordimeform) 0,0-diethyl-0-(2-isopropyl-4-methyl-6-pyrimidyl)-phosphorothioate (diazinon) octachlorocamphene (toxaphene) 0-ethyl 0-p-nitrophenyl phenylphosphonothioate (EPN) cyano(3-pheno~yphenyl)-methyl 4-chloro-a-(l-methylethyl)benzeneacetate (Pydrin~, fenvalerate) (3-pheno2yphenyl)methyl (+)-cis,trans-3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarbo~ylate (permethrin) dimethyl N,N'-[thiobis(N-methylimmo)carbonylo~y]]-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-pheno~yphenyl)methyl 4-(difluoromethoxy)-a-(methylethyl)benzeneacetate tPay-Off~, flucythrinate) 0,0-diethyl-0-(3,5,6-trichloro-2-pyridyl)phosphoro-thioate (Dursban~, chlorpyrifos) 0,0-dimethyl-S-[(4-o~o-1,2,3-benzotriazin-3-(4H)-yl)methyl~phosphorodithioate (Guthion~, azinphos-methyl) 5,6-dimethyl-2-dimethylamino-4-pyrimidinyl dimethyl carbamate (Pirimor~, pirimicarb) S-(N-formyl-N-methylcarbamoylmethyl)-0,0-dimethyl phosphorodithioate (Anthio~, formothion~

,,, ' . ,.. . . ~

~' - ' 3l 2 ~1 6! 7 3~ 4 2 S-2-(ethylthioethyl)-O,O-dimethyl phosphiorothioate ~demeton-S-methyl) ~-cyano-3-phenoxybenzyl cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carbo~ylate (Decis~, deltamethrin) cyano(3-pheno~yphenyl)methyl ester of N-(2-chloro-~-trifluoromethylphenyl)alanine (Mavrik~, fluvalinate).

The following E~amples illustrate the invention.

~XAMPLES
First Antibod~ OPtimization for Incr ased Sensitivi~ to ~etsulfuron meth~l The following definitions are used in the Examples:
"% CV" is the coe~ficient of variation, which is defined as:
Standard Deviation % CV = X 100, Mean "ma" refers to milli-absorbance units.
nWashi~g~ 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 polyo~yethylene sorbitan esters available .rom the Sigma Chemical Company.
Tissuemizer~ is a homogenizer manufactured by the TekMar Corporation.
O.D. refers to optical density.

: . , , - . ~ ,. : , . . .

.. , . ~ . . . . - . . -. ..

. : , .
- . :.

WO91/05259 PCT/US90/05424 _ 2~7342 ` 32 A. The coating conjugate coating step was conducted by dispensing 200 ~1 of coating conjugate at a concentration of 0.1 ~g/ml in 0.1 M NaHCO3, pH
9.4, in each well of a 96-well, flat-bottomed microwell plate and incubating at 4C overnight.
This time was chosen for convenience. Two hours at room temperature produces the same results.
A concentration of 0.1 yg/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 pest.icide 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 (~LH) 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.

.
, WO91/0525~ PCT/US90/05424 33 2 0~ 7,34 2 B. Micro~late Wa$hina A wash solution, consisting of phosphate-bufered saline (PBS) which contains 0.12 M NaCl, 2.7mM KCl, 10 mM phosphate buffer at a pH of 7.5 at 25C, and 0.05So Tween-20~, was used to remove e~cess coating conjugate from each well. Then, each well is filled with 200 ~1 of 3% BSA in PBS solution at room temperature and incubated for 2 hours to block the remaining sites. E~cess blocking solution was then removed by washing and the plates are shaken to remove any droplets of moisture and stored at 4C in sealed plastic bags.
C. Flrst AntibodY Reaqent The first antibody used in this assay was whole rabbit antiserum raised against an immunogen conjugate consisting of a carbo~ylic derivative of metsulfuron methyl, Compound (4), which was covalently coupled to the protein keyhole limpet hemocyanin (KLH). The antiserum, diluted in PBS/0.5%
~SA, comprises the first antibody reagent. Antihody dilutions of 1:100, l:lZ5, 1:165, 1:250 and Z5 1:500 X 103 were made and standard curves were generated with each of these antibody dilutions.
: ,. ..

HO2C~02NHCNH~ N
co2c~3 oCH~

Conpound (4) .. ~. .. . ..
.
:. . :

~ ' '. . , :
. .

WO91/0~2S9 PCT/U~9~/0~42~_~
20673~2 34 D. La~eled Second Antibody~Enzy~ Con~uaate 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 10 range is l:5,000 to l:5p,000 for enzyme immunoassays using PNPP. In the standard ELISA assay, a l:5,000 (.0012 mg/ml) dilution was made with PBS/0.5% ~SA.

E. ~nzyme Substrate Reaqent The enzyme substrate was formed from l 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 NaN2, lO0 mg of MgCl26H2O; l M HCl was added until the pH
is 9.8. The total volume was made up to l liter with water and stored at room temperature in an amber bottle. The p-nitrophenyl phosphate is commercially available.

" ,-F. As~ay Procedure The assay was performed at room temperature and consisted of l.0 ml of sample plus 0.8 ml PB5 plus 0.2 ml of lO X PBS/1% ~SA for a total volume of 2.0 ml. The first antibody, 50 ~l, was added to each sample. Standards were prepared similarly. The standards were 0, 0.005, O.OlO, 0.025, 0.050 ng/ml.
Each standard curve contained the antibody in one of the dilutions as described in C. After vorte~-mi~ing and one hour preincubation, 200 ~l of each reaction mi~ture was addPd to microplate wells in triplicate.
After one hour incubation on the microplate, the .~

W~91/05259 PCT~US90/05424 ~ 20~7342 plate was washed and 200 ~1 of second antibody-enzyme conjugate was added and incubated for 1 hour on the microplate. The microplate was washed again and 200 ~1 of substrate solution was added. A microplate reader was used to measure the absorbance at 405 nm 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:

Negati~e Control Absorba~ce -Standard Absorbance X 100 = ~ Inhibition.
Negative Co~trol Absorbance zO where the negative control absorbance is the ma~imum O.D. measured in the absence of antigen and the ~
standard absorbance is the O.D. obtained at the same -time (as the negative co~trol) with a known concentration of antigen. Thus, 0% inhibition indicates no analyte is present. When the percent inhibition e~ceeds 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.

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- . . . . :
.

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WO91/05259 PCT/US~0/05424 _ 2~73~2 36 First ~Picogr~l Antibody 5 lQ 25 _so 5 Dilution X 103 P~rcç~ ki~iQn~
l:loo h 28 5Z
1:125 ~ 25 42 61 1:165 * 26 ~4 65 1:250 * 30 56 7~
1:500 29 47 67 75 ~<15~. At least 15% inhibition is necessary to obtain accurate measurements and distinguish from neyative 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 103 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.

Fi~t An~ibody O~imization for Increa~ed SensitivitY to Cklorimu~on ethYl ~c ~
02~H
CO
NH

~1 Conpo und ( ~ ) WO91/05~59 PCT/US90/0~24 37 20~73~2 S A. Antiqen Coniu~at~ ~oatinq a~d_Blockinq of Microplates First, 200 ~l of antigen-protein conjugate at a protein concentration of O~l ~g~ml in O.l M NaHCO3, pH 9.4 was placed in each well of a 96-well flat-bottomed microplate and incubated overnight at 4C. 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 l.
The concentration of the coating conjugate was selected in a checkerboard assay as described in Example l.

B. First Antibody Reaqen~

HO~C ~C30ZEt ~

Cl OCH3 Co~po~nd C5) The ~irst antibody used in this assay was whole rabbit antiserum raised against an immunogen . .. . . ~ , . .
' ~ ': ' . .
.
- . . ~
, :. .

WO91/0~2~9 PCT/US90/0542 2 0~ 3 ~2 38 conjugate consisting of a carbo~ylic acid derivative of chlorimuron ethyl, Compound (5), which was covalently coupled to the protein keyhole limpet hemocyanin (KLH). The antis2rum diluted in PBS/0.5%
BSA comprises the first antibody r~eagent.

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

D. Enzyme Substrate Reaqent This was prepared as in the metsulfuron methyl assay of Example l.

E. Assay Procedure The assay was performed substantially as in the metsulfuron methyl assay (Example l). Standards were prepared at 2.5, 5, lO, and 50 picogram/ml of chlorimuron ethyl by adding 20 ~l of an appropriate concentration stock solution to l.8 ml of water 2S only. To each standard was added 200 ~l of 10%
PBS/1% ~SA and lO ~l of one of the four first antibody reagent dilutions, then each tube was vortex-mixed and incu~ated at room temperature for one hour.
F. Re~lts The following data demonstrates the increased sensitivity of the assay resulting from the decreased antibody concentration.

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W091/0~259 PCT/US90/05424 20~73~2 39 ~ ~

FiDal ( Picoqr~n~ml ) Antibody 2 . 55 10 50 5Dilut~ X 10;1 Per~at IIIhibition*
64 ~ 20 27 61 ' 128 ~ 21 33 71 320 24 39 5~ 77 640 23 45 57 72 :.

*c15~. At least 15% inhibition is necessary to obtain accurate measurements and distinguish from negative control.
These results demonstrate the improved sensitivity obtained by diluting first antibody.
15 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 ~econd AntibodY-Alkaline ;Phosph~tase Con~u~ç_ÇonGentratiQn ..
A. The ELISA procedure was e~actly as described above with the e~ception that first antibody dilution was 1:500 X 103 in the assay in all cases; and second antibody-enzyme conjugate concentration was 1.2, 2.4 or 4.8 ~g/ml. The concentration of 1.2 ~g/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 th~
percent inhibition for each of the standards with the three concentrations of second antibody-enzyme , .

.:
" " ~ , " ~

WO91~05259 PCT/US90/05424 20673~ q conjugate but a significant decrease in time for the assay to develop was seen with inc:reased concentration.

Substrate Conjugate Pi~oarams/r~L____ Incubation mg/ml 2.~ 5 lQ ~25 50 Time (min) ~ERCE~_INHIl~ITI~N
10 Standard E~ISA 1.2 17 31 46 64 72 155 E~. 1 2.4 16 28 42 G4 72 l:L0 E~. 2 4.8 19 36 48 65 73 80 Further increases in second antibody-enzyrne conjugate did not result in any further decreases in substrate incubation time.
OPtimization of A~say Time by Increasing Second Antibody Concen~ration A. Reaaents_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 ~g/ml conjugate concentrations were used in the assays.

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.
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wosl/o52~9 PCT/US90/05424 20673~2 , B. B~l~
The following data show the decreased substrate incubation time resulting from the increased concentration of labeled antibody-enzyme conjugate. l' ConjugateSubstrate Incuhation Time Micro~rams~ml minu~es Control 1.2 156 Ex. 3 2.4 124 E~. 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.

A. Precision The E~ISA 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 1:500,003, the standard and high sensitivity assay, respectively. The labeled second antibody enzyme conjugate concentration was at 1.2 ~g/ml for the standard assay and at 4.8 ~g/ml for the high sensitivity assay.
High Standard Sensitivity Found Found 30Metsulfuron-Methyl 1:100,000 1:500,000 Picoqrams/ml ma X % Cv ma X % Cv - 2127 1.52115 1.4 (EX. 5) 5 1970 2.91353 1.3 (Ex. 6) 10 1697 3.2 982 1.6 , ... .
. . ....

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WO91/0~25~ PCTIUS90/05424 2~67~2 42 This result clearly demonstrates that the precision of the assay, as indicated by % CV, actually improves at lower concentrations in the high-sensiti~ity format described in this invention.
Under these conditions, at higher sulfonylurea concentrations of 25, 50 and lO0 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 lO0 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 lO to 50 pg/ml in soil of the follbwing types (after e~traction into an aqueous medium): I'AMA, pH

- 6.5 to 7.0, organic matter = 2.6%; SASSAF~AS, pH
6.0, organic matter = 0.1%; and FAXGO, pH = 7.6, organic matter , 6%.
Likewise, metsulfuron methyl was measured at about lO0 pg/ml (after e~traction into an aqueous medium) in corn forage and corn stover.

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Claims (17)

What 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;
(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.

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 antihody 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.