Note: Descriptions are shown in the official language in which they were submitted.
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.
, . . . . . .. .
.:: , . ' ; ~ '
.: .' ' ,. ' ~ ,
.. : . : .
.
.:
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:
OC~
O,\,O ~ ~ IH
[
~N~
CH~
~ ~NH ~ C~`cco~ ~Z~
H~c~ CH3
.
: . .
~/0 91/05t~9 PCI/l~S90/05424
7 `2 67 42
~ `COOH
OOC~19
~ HOCC~[~S`NHJ~NH~OCH3
CC)OCH~
HOOC~W~ 5)
COOC~12CH,
.. . .
;,, ' ' '
WO 91/05~59 P~US90/05424 .~
~Q6-73~2 8
. .,
OCH( CH3~)2'
10~ ~ `NH ~~ ~ ~l OCH~C~13~z (
HOOC
7)
H~C
-
Cl
o~\~o,Ll~n3 t0~ ~:
3 5 . ,
' ` . , ' ' ' ' .~ ', ~ , ' . ' ' .
' ' ' : . ' ' ' ' . , , : .
WO 91/05259 PCI'/US90/05424
20~7342
c~3
~ J~WH,
CH,
[~f ~J~H
COOH
N}~CH3
QOC~ c~ HzCH3
OOCH3
`
. ' ` ' ~
. ' ' ,:
WO 91/05259 PCT'/I IS90/05424 _ .
20~342 lo
~, : '.
~' ~ ~ ~12) ; ~
ooc}~ ~ '
.
OCH3
2 0 ~H~SO, NHCMH~(N ( 1 3 )
02cH3 OCH2CO2H
oc~ : :.; ~ .
~I~,SO NE}CON~ U ~I 4~ .
~C~CH3
CO,~ ,
WO91/05259 PCT/US90/05424
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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W O 91/05259 PC~r/US90/05424
., . .`,` .i . .
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
~. . . ..
':~
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
: . . . - . .
.~ , .
,
. . . . . ..
- . ,
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
,
.
"~ :- ...
.. : ,
: ' - ' .
WO91/05259 PCT/US90/05424
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
.
.
- . .
. ' : . ' ;'' ,: , ' ',
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
., . . -
. . . . . . .
,. , .. . . . . . :
:, , . , ~ . . .; ~ ~ .:
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
- - , . , . . . . . ~ . : . .
' . ' ' ..... . ~ :.' ~.... .
J
' - ` . ~ ~ .
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
.
,~ ,
~,
,,.. ; .. , , , , .. ~ . . ., . ,", ... .. ..
.: , . :, ~ , , ,, : ,
.. . - . . . - ,, . , "
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
23 2067342
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~
,,, '
. ,.. . . ~
~' - '
WO91/052S9 PCT/US90/05424
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.
: ,. ..
O CH3
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.
', ': ' , ~', ' ` :
- . . . . :
.
'': ' .: ' ~`
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
CH30
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
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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
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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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wosl/o52~9 PCT/US90/05424
20673~2
41
,
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.
EXAMPLES S AND 6
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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