Note: Descriptions are shown in the official language in which they were submitted.
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The present invention generally relates to assays for determining levels of
S compounds) in a liquid medium. More particularly this invention relates to
methods
for determining the level of giyphosate in water at concentrations as low as
the parts
per billion (ppb) level.
The global use of glyphosate-containing herbicides creates a continuous need
for the measurement of glyphosate in a variety of matrices. Current methods
for
glyphosate analysis are tedious and require sophisticated equipment.
Glyphosate was introduced by Monsanto Company as a new herbicide in 1974
and is now sold globally under numerous trademarks, including RoundupTM
herbicide.
Chemically, glyphosate (N-phosphonomethylglycine; C3H$NOSP; M.W.,169.1) is a
small zwitterionic amino acid derivative that poses unique problems in the
development of analytical methods. Due to the small size of the analyte and
its
insolubility in common organic solvents, most analytical methods for
glyphosate
involve extensive sample cleanup, derivatization, and separation on gas or
liquid
chromatography columns. These chromatographic techniques are slow and
cumbersome and require sophisticated equipment. An excellent overview of
current
analytical methods for detecting glyphosate can be found in Franz, et al.,
Glyphosate:
A Unique Global Herbicide, American Chemical Society Monograph 189, pp 80-97,
Washington, DC., 1997.
In 1974, Congress passed the Safe Drinking Water Act. This law requires the
Environmental Protection Agency of the United States Government, the EPA, to
determine safe levels in drinking water of chemicals that do, or may, cause
health
problems. These levels are called Maximum Contaminant Level Goals (MCLG). the
MCLG for glyphosate has been set at 7 ppb because the EPA believes this level
would not cause any of the known or suspected potential health problems. In
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2
addition, the EPA believes, given present technology and resources, this is
the lowest
level to which water systems can reasonably be required to remove this
contaminant
from drinking water supplies.
Therefore, the development of a simple, rapid, cost-efficient immunoassay
method for glyphosate is highly desirable. However, a major challenge to
developing
a successful immunoassay for a small analyte, such as glyphosate, is the
difficulty of
generating analyte-specific antibodies having sufficiently high affinity
toward the
analyte to be useful in such assay protocols as a competition enzyme-linked
immunosorbent assay (ELISA).
Since small molecules are "overlooked" by the immune system, it is common
to enhance the immunogenicity of low molecular weight analytes (haptens) (100-
200
Daltons) by covalently conjugating the analyte, or its analogue, to an
immunogeruc
Garner protein. However, it is now widely realized that antibodies raised
against such
conjugates rarely have sufficient affinity for the unconjugated hapten, which
is the
analyte of interest, to be useful for most immunoanalytical purposes. It is
believed
that the surface area of the anaiyte is insufficient for optimal interaction
with the
antibody combining site of the antibody molecule (Chappey et al., Journal of
Immunological Methods ,1219-225, 1994). Generally, this phenomenon is due to
the antibody having been originally selected to recognize the hapten as well
as the
linkage arm binding the hapten to the carrier protein in the immunizing
conjugate.
Thus, the design for the synthesis of protein-hapten conjugates has a profound
influence on the specificity and sensitivity of an assay in which the antibody
is used.
One method for overcoming this problem is to enhance affinity for a small
analyte by chemically increasing the size of a hapten in the sample to be
tested
through formation of chemical derivatives. U.S. Patent No 4,818,683 to Morel
and
Delaage (1989) discloses an immunoassay method for monoarnines based on
chemical conversion in a sample intended for analysis, of a monoamine analyte,
such
as histamine, into a chemical derivative of higher molecular weight. Succinyl
glycinamide (SGA) derivatives of the monoamine analyte, formed by acyladon,
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3
incorporate the SGA moiety into the analyte in a test sample prior to assay of
the
sample to detect the presence of the analyte.
However, this prior art method is synthetically complicated since it requires
reaction of samples with a novel acylation reagent, such as N-
hydroxysuccinimide-
ester-succinyl-glycinamide, to form a chemical derivative of the monoamine
analyte
in the sample prior to testing. Further, the success of this method requires a
procedure
that is slightly more complicated than the standard procedure for synthesis of
the
immunizing conjugate used to raise antibodies to be employed in the
immunoassay.
The immunizing conjugate is also a succinyl glycinamide (SGA) derivative
formed by
acylation of the monoamine analyte and purification of the derivatized analyte
is
required prior to conjugation of the derivative to a carrier protein to
increase the
immunogerucity of the conjugate.
For nearly four decades, immunoassay procedures have provided sensitive
diagnostic tools for the in vitro detection of a variety of antigens,
including those
associated with disease or other physical conditions of clinical significance.
These
procedures are now being used at an accelerated pace for the detection and
quantitation of pesticides in various biological and environmental samples.
There are
many variations on the ways in which immunoassays can be performed. Three
classes of immunoassays are commonly used, the antibody capture assay, the
antigen
capture assay, and the two-antibody sandwich assay. In an antibody capture
assay,
the antigen is attached to a solid support, and labeled antibody is allowed to
bind.
After washing, the assay is quantitated by measuring the amount of antibody
retained
on the solid support. In an antigen capture assay, the antibody is attached to
a solid
support, and the labeled antigen is allowed to bind. The unbound components
are
removed by washing, and the assay is quantitated by measuring the amount of
antigen
that is bound. 1n a two-antibody sandwich assay, one antibody is bound to a
solid
support, and the antigen is allowed to bind to this first antibody. The assay
is
quantitated by measuring the amount of a labeled second antibody that can bind
to the
antigen. Generally the sandwich assay is not applicable to a small analyte
such as
glyphosate because of its inability to serve as a binding partner for both of
the
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4
antibodies simultaneously. The general procedures and rationale for selecting
one
type of assay over another are well known in the art and are summarized in
Harlow
and Lane, Antibodies: A Laborator<r Manual, Cold Spring Harbor Laboratory, New
York, 1988, Chapter 14, which is incorporated herein by reference).
Heterogeneous assays use a polyclonal antibody preparation bound to the solid
phase. In these assays, a solution of labeled antigen is allowed to compete
directly for
the solid phase antibody with antigen in the sample being analyzed.
Alternatively, a
solution of labeled antigen can be added to the antibody in a sequential
process. The
extent to which the labeled antigen is bound to the solid phase, or is
detected in the
liquid phase, can be used as a measure of the presence and quantity of antigen
in the
sample being analyzed. Immunoassay procedures modified to use monoclonal
antibodies are also known in the art. For example, U.S. Pat. No. 4,376,110
describes
two-site immunometric assays using pairs of monoclonal antibodies, one bound
to a
solid phase and the other labeled to permit detection. The use of monoclonal
antibody
pairs which recognize different epitopic sites on an antigen has made it
possible to
conduct simultaneous immunometric assays in which the antigen and labeled
antibody
incubations do not require the intermediate washing steps of prior processes.
In view of the above state of the art and interest in developing assays for
detection of concentrations of low molecular weight molecules, there is a need
in the
art for new and better methods for routine analysis of glyphosate utilizing a
simple,
high throughput immunoassay format.
The present invention overcomes many of the problems in the art by providing
methods for simple, inexpensive and high throughput assay of the widely used
herbicide, glyphosate. One object of the present invention is to provide
analyte-specific antibodies against glyphosate and chemically similar
compounds for
use in immunoassays, such as ELISA, through a simple approach to immunogen
preparation that does not require derivatization of the hapten prior to its
conjugation
to an immunogenic protein.
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A further object of the present invention is to provide a highly specific and
sensitive (ng/ml level) linker-assisted immunoassay method for glyphosate in
test
samples, such as drinking water, extracts of soils, and the like.
These objects of the invention are met by providing (i) strategically designed
5 protein-hapten conjugates for use as immunogens and as solid-phase-coating
antigens
in ELISA-based procedures, and (ii) a simple affinity-enhancing analyte
derivatization procedure that enhances assay sensitivity, for example,
providing an
increase in assay sensitivity of up to 104-fold or greater. Invention
immunoassay
methods) require a simple pre-assay derivatization step during which the
analyte is
covalently attached to a linker moiety, such as glutaric acid or succinic
acid, and use
commercially available, inexpensive, and relatively stable reagents.
Therefore, in one embodiment according to the present invention, there are
provided methods for obtaining an anti-glyphosate antibody. Invention antibody
production methods) comprise preparing an immunogenic conjugate by covalently
coupling glyphosate, a derivative containing at least two ionizable acidic
groups, or a
salt thereof, directly to a first carrier molecule, immunizing a susceptible
host at
variable intervals with the conjugate; and obtaining the antibody from the
host. When
the hapten is a derivative containing at least two ionizable acidic groups, or
salts)
thereof, the coupling step is conducted under conditions selected to preserve
the
chemical identity of the at least two ionizable acidic groups in the
derivative.
In another embodiment according to the present invention, there are provided
linker-assisted immunoassay methods for the detection of glyphosate, or a salt
thereof, in a test sample. Invention linker-assisted immunoassay methods)
comprise
reacting the test sample with a linker having an activated carboxylic group to
obtain
an analyte-linker conjugate, contacting the reacted test sample with at least
one
invention anti-glyphosate antibody, further contacting the test sample
containing the
analyte-linker conjugate with a solid phase having immobilized thereon a solid
phase
coating conjugate comprising a second carrier molecule covalently coupled to
glyphosate, a derivative containing at least two ionizable acidic groups, or a
salt
thereof, removing unbound components from the solid phase, and detecting the
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6
presence of bound anti-glyphosate antibody. The amount of bound antibody is
inversely related to the amount of glyphosate, or a salt thereof, in the test
sample. The
carrier molecule in the coating conjugate, however, is not identical to (i.e.,
different
than) the carrier molecule in the immunogenic conjugate used to obtain the
anti-glyphosate antibody. When the hapten is a glyphosate derivative having at
least
two ionizable acidic groups in the derivative, the coupling step is performed
under
conditions selected to preserve the chemical identity of the at least two
ionizable
acidic groups in the derivative.
In another embodiment according to the present invention, there are provided
test kits) for the immunochemical detection of glyphosate, or a salt thereof,
in a test
sample. Invention test kit{s) comprise a solid phase and at least one
invention
anti-glyphosate antibody, which is bound, or can be bound, to the solid phase.
The
test kit may further optionally comprise such additional reagents as a labeled
hapten
conjugate that binds to the antibody to create a labeled anti-glyphosate
antibody, and a
linker such as, for example, aspartic, glutamic, succinic, glutaric, adipic,
N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic, or poly-glutamic acids.
Figure 1 is a graph showing the results of comparative ELISA tests for
determining the glyphosate concentration (ng/ml) in an aqueous sample by the
invention linker-assisted immunoassay methods) (-~-) and by standard ELISA
technology (-1-) . The tests were conducted in parallel using identical plates
and
identical solutions containing antibodies raised against a glyphosine-porcine
thyroglobulin (TG) conjugate. The results are shown as relative absorbance at
450
nm. Relative absorbance was obtained by dividing the mean optical density
value for
each standard point by the mean optical density value of a standard containing
no
analyte.
Figure 2 is a graph showing the results of parallel comparative inhibition
ELISA tests for determining the glyphosate concentration (ng/ml) in an aqueous
sample using anti-TG-glyphosine antibody (-~-) or anti-TG-glyphosate (-1-)
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7
antibody in linker-assisted ELISA tests wherein the analyte is derivatized by
reacting
it with glutaric anhydride. The results are shown as relative absorbance at
450 nm.
The present invention is based upon the discovery that the sensitivity of an
immunoassay, such as a competition ELISA wherein the analyte is a low
molecular
weight molecule, can be enhanced if the analyte in the sample is conjugated
prior to
assay with a linker molecule such that the analyte conjugate in the sample
mimics the
immunorespvnsive portion of the hapten-protein conjugate used to raise
antibodies
prepared for use in the assay.
The glyphosate molecule contains three functional moieties (i.e., carboxylate,
secondary amine, and phosphonate) separated by two methylene groups. Under
physiological conditions, the phosphonate and carboxylate of glyphosate are
negatively charged, and hence would serve as immuno-dominant groups for this
hapten. The conjugation procedures used in carrying out this invention are
designed
, to ensure that the chemical functionality of both of these groups is
preserved during
the coupling of this hapten to a Garner protein, when an immunostimulatory
conjugate
is prepared to raise antibodies to be used in an immunoassay and when a
coating
conjugate or a signal-generating conjugate (for example, horseradish
peroxidase-
glyphosate) is prepared for use in the immunoassay.
In invention immunoassay method(s), analyte in the test sample is derivatized
so as to make the derivative more closely mimic the epitopic site to which
antibodies
raised against the immunogenic conjugate will bind with enhanced affinity. In
derivatizing analyte in the test sample prior to testing, care should be taken
to assure
that the ionic character of negatively charged groups in the analyte are
preserved in
the derivative.
Therefore, in one embodiment of the present invention, there are provided
methods) for obtaining an anti-glyphosate antibody. Invention antibody-
producing
methods) comprise preparing an immunogenic conjugate by covalently coupling
glyphosate, a derivative containing at least two ionizable acidic groups, or a
salt
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8
thereof, directly to a carrier molecule, immunizing a susceptible host at
variable
intervals with the conjugate; and obtaining the anti-glyphosate antibody from
the host.
Derivatives of glyphosate that contain at least two ionizable acidic groups,
or salts
thereof, can substitute for glyphosate in invention immunogenic conjugate(s).
The
coupling step in invention anti-glyphosate producing methods) is performed
under
conditions selected to preserve the chemical identity of the at least two
ionizable
acidic groups in glyphosate or its derivative.
More particularly, when the immunogenic conjugate is obtained by attaching a
carrier molecule to glyphosate, or a salt thereof, the carrier molecule
preferably has
free carboxyl groups, and imide bonds are formed between the carboxyl groups
of the
carrier molecule and the secondary amino group of glyphosate to form an
immunogenic conjugate having two negatively charged groups that mimic the
negative charges of free glyphosate. In these circumstances, the coupling is
generally
achieved in a two-step process, by first activating the carboxyl groups on the
carrier
protein using an activating agent, such as 1-ethyl-3-(3-diaminopropyi)
carbodiimide
hydrochloride (EDC), followed by a nucleophilic reaction with glyphosate, or a
salt
thereof. Generally the glyphosate or glyphosate derivative contributes a
secondary
amino or carboxylic group, and the carrier molecule contributes a carboxylic
or
primary amino group toward formation of the linkage, (e.g., via an active
ester or a
water soluble carbodiimide). The preferred glyphosate salt is a sodium salt.
For example, the carboxyl groups on the carrier molecule can be activated
with EDC for about 2-5 minutes at pH of about 5, followed by a nucleophilic
reaction
with glyphosate at alkaline pH in the presence of a molar excess (over EDC) of
phosphate. The excess phosphate in this reaction serves to quench EDC, thereby
preventing it from activating the carboxylic group of glyphosate. The acidic
pH is
preferably maintained between about 4 and about 6, and the alkaline pH is
preferably
maintained between about 7.5 and 9.5.
An alternative strategy for preparation of an immunogenic conjugate that will
yield anti-glyphosate antibodies comprises the use of a glyphosate derivative
having
two ionizable acidic groups, as a surrogate for glyphosate. Wben a glyphosate
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9
derivative having at least two ionizable acidic groups, or a salt thereof, is
used to
obtain invention anti-glyphosate antibodies, coupling is preferably achieved
in a two-
step process, by first performing carbodiimide-mediated activation of the
derivative
under acidic pH conditions, and then reacting the activated carboxylic group
of the
derivative with the amino groups of the carrier molecule under alkaline pH
conditions. The acidic pH is preferably maintained between about 4 and about
6, and
the alkaline pH is preferably maintained between about 7.5 and 9.5.
For example, glyphosine (N,N-bis(phosphonomethyl)-glycine) is a glyphosate
derivative containing one carboxymethyl and two phosphonomethyl groups
attached
to a tertiary amino group. In the invention method, EDC-mediated coupling of
the
carboxyl group of glyphosine to the lysine groups of a carrier protein, such
as porcine
thyroglobulin (TG), leaves two negatively charged phosphonic acid groups to
mimic
the negative charges of free glyphosate. Additional non-limiting examples of
suitable
glyphosate derivatives having at least two ionizable acidic groups that can be
used as
1 S the hapten in preparation of invention immunogenic conjugates) include
N-phosphonomethyliminodiacetic acid, iminodiacetic acid, N,N-
bis(phosphonomethyl) amine, and the like.
Non-limiting examples of carrier molecules useful in preparation of invention
immunogenic conjugates) include porcine thyroglobulin, bovine serum albumin,
human serum albumin, ovalbumin, keyhole limpet hemocyanin, and the like. The
presently preferred carrier molecules are proteins, such as porcine
thyroglobulin (TG)
or bovine serum albumin (BSA). The preferred molecular weight range for the
carrier
molecule is from about 100,000 to about 10,000,000.
Antibodies used in invention assays) can be polyclonal, monoclonal, or a
functionally active fragment thereof. Mono- or poly-clonal antibodies to
glyphosate,
its salts, and glyphosate derivatives, are raised in appropriate host animals
by
immunization with invention immunogenic conjugates) using conventional
techniques as are known in the art.
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The preparation of monoclonal antibodies is disclosed, for example, by Kohler
and Milstein, Nature 2:495-7, 1975; and Harlow et al., in: Antibodies: a
Laboratory
page 726 (Cold Spring Harbor Pub., 1988), which are hereby incorporated by
reference. Briefly, monoclonal antibodies can be obtained by injecting mice,
or other
5 small mammals, such as rabbits, with a composition comprising an invention
immunogenic conjugate whose preparation is disclosed above, verifying the
presence
of antibody production by removing a serum sample, removing the spleen to
obtain B
lymphocytes, fusing the B lymphocytes with myeloma cells to produce
hybridomas,
cloning the hybridornas, selecting positive clones that produce antibodies to
the
10 antigen, and isolating the antibodies from the hybridoma cultures.
Monoclonal
antibodies can be isolated and purified from hybridoma cultures by a variety
of well-
established techniques. Such isolation techniques include affinity
chromatography
with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Barnes et al., Purification of
Itnmunoglobuiin G
(IgG), in: Methods in Mol .Biol., ,~Qi 79-104,1992). Antibodies of the present
invention may also be derived from subhuman primate antibodies. General
techniques for raising antibodies in baboons can be found, for example, in
Goldenberg
et al., International Patent Publication WO 91/11465 (1991) and Losman et al.,
Int. J.
Cancer, 4:310-314, 1990.
It is also possible to use anti-idiotype technology to produce monoclonal
antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal
antibody made to a first monoclonal antibody will have a binding domain in the
hypervariable region which is the "image" of the epitope bound by the first
monoclonal antibody.
The term "antibody" as used in this invention includes intact molecules as
well
as functional fragments thereof, such as Fab, F(ab')Z, and Fv that are capable
of
binding glyphosate, or a salt thereof, especially after the glyphosate or salt
thereof has
been derivatized with a linker molecule as disclosed herein. These fiuictional
antibody
fragments are defined as follows:
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(1) Fab, the fragment which contains a monovalent antigen-binding
fragment of an antibody molecule, can be produced by digestion of whole
antibody
with the enzyme papain to yield an intact light chain and a portion of one
heavy chain;
(2) Fab', the fragment of an antibody molecule that can be obtained by
treating whole antibody with pepsin, followed by reduction, to yield an intact
light
chain and a portion of the heavy chain; two Fab' fragments are obtained per
antibody
molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by treating
whole antibody with the enzyme pepsin without subsequent reduction; F(ab')Z is
a
dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the
variable region of the light chain and the variable region of the heavy chain
expressed
as two chains; and
(5) Single chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain and the variable region of
the heavy
chain, linked by a suitable polypeptide linker as a genetically fused single
chain
molecule.
Methods of making these fragments are known in the art. (See for example,
Harlow and Lane, ,f~tltibodies: A Lahorat~ rv Manual, Cold Spring Harbor
Laboratory,
New York, 1988, incorporated herein by reference). As used in this invention,
the
term "epitope" means any antigenic determinant on an antigen to which the
paratope
of an antibody binds. Epitopic determinants usually consist of chemically
active
surface groupings of molecules such as amino acids or carbohydrate side chains
and
usually have specific three dimensional structural characteristics, as well as
specific
charge characteristics.
Antibody fragments according to the present invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E. coli of DNA
encoding
the fragment. Antibody fragments can be obtained by pepsin or papain digestion
of
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12
whole antibodies by conventional methods. For example, antibody fragments can
be
produced by enzymatic cleavage of antibodies with pepsin to provide a SS
fragment
denoted F(ab')2. This fragment can be further cleaved using a thiol reducing
agent,
and optionally a blocking group for the sulfhydryl groups resulting from
cleavage of
disulfide linkages, to produce 3.SS Fab' monovalent fragments. Alternatively,
an
enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an
Fc
fragment directly. These methods are described, for example, by Goldenberg,
U.S.
Patent Nos. 4,036,945 and 4,331,647, and references contained therein, which
patents
are hereby incorporated by reference in their entirety. See also Porter, R.R.,
Biochem.
14 J., Z~: 119-126, 1959. Other methods of cleaving antibodies, such as
separation of
heavy chains to form monovalent light-heavy chain fragments, further cleavage
of
fragments, or other enzymatic, chemical, or genetic techniques may also be
used, so
long as the fragments bind to the antigen that is recognized by the intact
antibody.
Fv fragments comprise an association of VH and VL chains. This association
may be noncovalent, as described in mbar et al., Proc. Nat'1 Acad. Sci. USA
~~:2659-
62, 1972. Alternatively, the variable chains can be linked by an
intermolecular
disulfide bond or cross-linked by chemicals such as glutaraldehyde.
Preferably, the
Fv fragments comprise VH and VL chains connected by a peptide linker. These
single-chain antigen binding proteins (sFv) are prepared by constructing a
structural
gene comprising DNA sequences encoding the VH and VL domains connected by an
oligonucleotide. The structural gene is inserted into an expression vector,
which is
subsequently introduced into a host cell such as E. coli. The recombinant host
cells
synthesize a single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing sFvs are described, for example, by Whitlow and
Filpula, Methods, 2: 97-105, 1991; Bird et al., Science ?x:423-426, 1988; Pack
et al.,
BiolTechnology 11:1271-77, 1993; and Ladner et al., U.S. Patent No. 4,946,778,
which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single
complementarity-determining region (CDR). CDR peptides ("minimal recognition
units' can be obtained by constructing genes encoding the CDR of an antibody
of
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13
interest. Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of antibody-producing
cells.
See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
In another embodiment according to the present invention, there are provided
methods for obtaining strategically designed protein-hapten conjugates for use
as
solid-phase coating conjugates in invention ELISA-based assays) for detection
of
glyphosate, or a salt thereof. Preparation of a solid phase coating conjugate
for use in
invention assays comprises covalently linking glyphosate, a derivative
containing at
least two ionizable acidic groups, or salts) thereof, to a carrier protein
that is different
than (i.e., not identical to) the Garner protein used in obtaining antibodies)
for use in
invention ELISA-based assay(s). For example, if the carrier protein used in
invention
immunogenic conjugates) to obtain anti-glyphosate antibodies) is
thyroglobulin, the
carrier protein used in the coating conjugate is not thyroglobulin, but is
selected from,
for example, bovine serum albumin or ovalbumin. However, the coating conjugate
mimics the ionic characteristics of the immunogenic conjugate to the extent
that
negatively charged groups in the glyphosate, a derivative containing at least
two
ionizable acidic groups, or salts) thereof, are preserved in the coating
conjugate. In
the practice of invention assay method(s), it is not necessary that the
glyphosate
derivative used in the coating conjugate be identical to a glyphosate
derivative used to
obtain invention immunogenic conjugate(s).
In general, glyphosate derivatives suitable for use in the preparation of
invention coating conjugates) can be chosen from N-phosphonomethylglycine
(with
the preferred linkage site being the secondary amino group thereof), N, N-
bis(phosphonomethyl) glycine (with the preferred linkage site being the
carboxyl
group thereof), N, N-bis(phosphonomethyl) amine (with the preferred linkage
site
being the secondary amino group thereof), N-phosphonomethyl-iminodiacetic acid
(with the preferred linkage site being at one of the two carboxyl groups
thereof),
iminodiacetic acid (with the preferred linkage site being the secondary amino
group
thereof), and the like.
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14
Preferred coating conjugates comprise N-phosphonomethylgiycine covalently
coupled to bovine serum albumin or N, N-bis(phosphonomethyl)glycine covalently
coupled to ovalbumin.
Invention coating conjugates) and antibody(ies) are successfully employed in
accordance with invention linker-assisted ELISA methods) for the detection of
glyphosate, or salts) thereof, in a test sample. Invention assay methods)
employ a
novel derivatization step wherein the test sample is reacted with a linker
having an
activated carboxylic group to conjugate the linker with glyphosate, or salts)
thereof,
in the test sample. Attachment of the linker to the glyphosate in the test
sample
enhances the affinity of invention anti-glyphosate antibodies) for the
glyphosate
therein.
'Therefore, in another embodiment according to the present invention, there
are
provided linker-assisted immunoassay methods) for the detection of glyphosate,
derivatives thereof containing at least two ionizable acidic groups, and
salts) thereof,
1 S in a test sample. Invention linker-assisted immunoassay methods) comprise:
(a) reacting the test sample with a linker having an activated carboxylic
group to obtain an analyte-linker conjugate in the test sample,
(b) contacting the reacted test sample with at least one invention
anti-glyphosate antibody,
(c) contacting the test sample containing the analyte-linker conjugate with
a solid phase having immobilized thereon a solid phase coating conjugate
comprising
a second carrier molecule covalently coupled to glyphosate, a derivative
containing at
least two ionizable acidic groups, or salts) thereof,
(d) removing unbound components from the solid phase, and
(e) detecting the presence of bound anti-glyphosate antibody,
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wherein the amount of bound antibody is inversely related to the amount of
the glyphosate, derivative thereof containing at least two ionizable acidic
groups, or
salts) thereof, in the test sample, and wherein the second carrier molecule in
the
coating conjugate is not identical to the carrier molecule in the immunogenic
5 conjugate used to obtain the anti-glyphosate antibody.
Preferably, the anti-glyphosate antibody is contacted with the test sample at
a
pH of about 7 to about 10, and the analyte-linker conjugate is formed at a pH
of about
7 to about 10 so that an activated carboxylic group on the linker becomes
attached to
glyphosate in the test sample via the secondary amine group thereof. Suitable
linkers
10 for reaction with the test sample include succinic, glutaric, adipic, N-
acetyl-aspartic,
N-acetyl glutamic, poly-aspartic, and poly-glutamic acids, succinic and
glutaric
anhydrides, and the like. Generally, the linker is covalently linked to the
secondary
amino group of glyphosate, thereby enhancing the affnity of the first antibody
for the
glyphosate.
15 Invention linker-assisted assay methods) may further comprise attaching a
detectable label to the anti-glyphosate antibody on the solid phase. For
example, the
anti-glyphosate antibody can be conjugated to biotin and the detecting will
comprise
contacting the anti-glyphosate antibody with an enzyme-labeled molecule that
binds
strongly to the biotin. Alternatively, invention immunoassay methods) can
further
comprise binding to the anti-glyphosate antibody on the solid phase a second
antibody
conjugated to a signal-generating agent, such as an enzyme, radioisotope,
chemiluminescent or fluorescent label, colored microbead, colloidal gold, and
the
like.
Radioisotopes suitable for use as a signal-generating agent in the practice of
invention immunoassay methods) include tritium, carbon 14, phosphorous 32,
iodine
125, iodine 131, and the like, which can be attached to an antibody by methods
well
known in the art. For example, l2sl can be attached to an antibody by
procedures such
as the chloramine-T procedure, or the lactoperoxidase procedure. These
techniques
plus others are discussed in H. Uan Vunakis and J. J. Langone, Editors,
Methods in
Enzymol~, Vol. 70, Part A, 1980, which is hereby incorporated by reference.
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Chromogenic labels suitable for use as a signal-generating agent in the
practice of invention immunoassay methods) include compounds that absorb light
in
the visible or ultraviolet wavelengths, and the like. Such compounds are
usually
dyestuffs and include quinoline dyes, triarylmethane dyes, phthaleins, insect
dyes, azo
dyes, anthraquinoid dyes, cyanine dyes, phenazoxonium dyes, and the like.
Fluorogenic compounds suitable for use as a signal-generating agent in the
practice of invention immunoassay methods) include those that emit light in
the
ultraviolet or visible wavelengkh subsequent to irradiation by light, and the
like. The
fluorogens can be employed by themselves or with quencher molecules. The
primary
fluorogens are those of the rhodamine, fluorescein, and umbelliferone
families. The
methods of conjugation and use of these and other fluorogens can be found in
the art.
See, for example, J. J. Langone, H. Van Vunakis et al., Methods in Enzymology,
Vol.
74, Part C, 1981, especially at page 3 through 105. For a representative
listing of
other suitable fluorogens, see Tom et al., U.S. Pat. No. 4,366,241, issued
Dec. 28,
1982, especially at column 28 and 29. For further examples, see also U.S. Pat.
No.
3,996,345, herein incorporated by reference.
Those skilled in the art will recognize that an enzyme-catalyzed signal system
is, in general, more sensitive than a non-enzymatic system. Thus, for use in
the
practice of the present invention, catalytic labels are the more sensitive non-
radioactive labels.
Catalytic labels are well known in the art and include single and dual
("channeled") enzymes such as alkaline phosphatase, horseradish peroxidase,
luciferase, (3-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase,
glucose-6-phosphate dehydrogenase, and the like. Examples of dual
("channeled")
catalytic systems include alkaline phosphatase and glucose oxidase using
glucose-6-
phosphate as the initial substrate. A second example of such a dual catalytic
system is
illustrated by the oxidation of glucose to hydrogen peroxide by glucose
oxidase,
which hydrogen peroxide would react with a leuco dye to produce a signal
generator.
A further discussion of catalytic systems can be found in Tom et al., U.S.
Pat. No.
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17
4,366,241, issued Dec. 28, 1982, herein incorporated by reference. (See
especially
columns 27 through 40.) Also, see Weng et al., U.S. Pat. No. 4,740,468, issued
Apr.
26, 1988, herein incorporated by reference, especially at columns 2 and
columns 6, 7,
and 8.
The procedures for coupling enzymes to antibodies are well known in the art.
Reagents used for this procedure include glutaraldehyde, p-toluene
diisocyanate,
various carbodiimide reagents, p-benzoquinone, m-periodate, N, Nl-o-
phenylenedimaleimide, and the like (see, for example, J. H. Kennedy et al.,
Clin.
Chim Acta 7Q:1 (1976)).
Preferred signal generating agents are horseradish peroxidase and alkaline
phosphatase.
Chemiluminescent labels are also applicable. See, for example, the labels
listed in C. L. Maier, U.S. Pat. No. 4,104,029, issued Aug. 1, 1978, herein
incorporated by reference.
The substrates for the catalytic systems include simple chromogens and
fluorogens such as para-nitrophenyl phosphate (PNPP), (3-D-glucose (plus
possibly a
suitable redox dye), homovanillic acid, o-dianisidine, bromocresol purple
powder, 4-
alkyl-umbelliferone, luminol, para.-dimethylaminoiophine, paramethoxylophine,
and
the like.
Depending on the nature of the label and catalytic signal producing system,
one would observe the signal by irradiating with light and observing the level
of
fluorescence: providing for a catalyst system to produce a dye, fluorescence,
or
chemiluminescence, where the dye could be observed visually or in a
spectrophotometer and the fluorescence could be observed visually or in a
fluorometer; or in the case of chemiluminescence or a radioactive label, by
employing
a radiation counter. Where the appropriate equipment is not available, it will
normally
be desirable to have a chromophore produced that results in a visible color.
Where
sophisticated equipment is involved, any of the techniques is applicable.
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The term "solid phase" as used herein means common supports used in
immunometric assays made fibm natural or synthetic materials. The solid phase
support is insoluble in water and can be rigid or non-rigid. Among such
supports are
filter paper, the wells of microtiter plates, filtering devices (e.g., glass
membranes),
S plastic beads (such as polystyrene beads), test tubes, strips, or (multiple)
test wells
made from polyethylene, polystyrene, polypropylene, nylon, nitrocellulose,
glass
microfibres, and the like. Also useful are particulate materials such as
agarose, cross-
linked dextran, and other polysaccharides.
The steps employed to remove the unbound components from the solid phase
for the various assay formats can be performed by methods known in the art.
Generally, a simple washing with buffer followed by filtration or aspiration
is
sufficient. After washing, it is sometimes appropriate, as with particulate
supports, to
centrifuge the support, to aspirate the washing liquid, add wash liquid again,
and
aspirate. For membrane and filters, additional washing with buffer may often
be
sufficient, preferably drawing the liquid through the membrane or filter by
applying
vacuum to the opposite side of the membrane or filter or contacting the
opposite side
of the filter or membrane with a liquid absorbing member that draws the liquid
through, for instance, by capillary action.
Moderate temperatures, such as room temperature, are normally employed for
carrying out the assay. Constant temperatures during the period of the
measurement
are generally required only if the assay is performed without comparison with
a
control sample. The temperatures for the determination will generally range
from
about 10°C to about-50°C, more usually from about 15°C to
about -45°C.
In another embodiment according to the present invention, there are provided
test kits) for the imrnunochemical detection of glyphosate, or salts) thereof,
in a test
sample. Invention test kits) comprise a solid phase, at least one invention
anti-glyphosate antibody, which antibody is bound, or can be bound, to the
solid
phase. Invention test kits) may further comprise a labeled hapten conjugate
that
binds to the anti-glyphosate antibodies) to create a labeled anti-glyphosate
antibody,
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and a linker selected from aspartic, glutamic, succinic, glutaric, adipic,
N-acetyl-glutamic, N-acetyl-aspartic, poly-aspartic and poly-glutamic acids,
and the
like.
Invention test kits) may fiuther be packaged in combination with
predetermined amounts of reagents for use in assaying glyphosate. Where an
enzyme
is the label, the reagents can include substrate for the enzyme or the
requisite
precursors for the substrate, including any additional substrates, enzymes,
and
cofactors and any reaction partner of the enzymatic product required to
provide the
detectable chromophore or fluorophore. In addition, other additives, such as
ancillary
reagents, may be included, for example, stabilizers, buffers, and the like.
The relative
amounts of the various reagents may vary widely, to provide for concentrations
in
solution of the reagents which substantially optimize the sensitivity and
specificity of
the assay. The reagents can be provided as dry powders, usually lyophilized,
including excipients, which on dissolution will provide for a reagent solution
having
1 S the appropriate concentrations fox performing the assay.
Invention test kits) are useful for determining the concentration of
glyphosate
or salts) thereof contained in such test samples taken from a variety of
sources, e.g., a
drinking water supply, an extract of an environmental specimen, an extract of
a plant
or soil specimen, an extract of a biological specimen, and the like. The
detection
sensitivity of the glyphosate in the sample is in the concentration range from
about
100 ppm to about 0.5 ppb.
The invention will now be described in greater detail by reference to the
following non-limiting examples.
EXAMPLE 1
All chemicals used in these Examples were reagent grade and commercially
available from sources such as Sigma (St. Louis, MO), Chem Service (West
Chester,
PA), Axnresco (Solon, OH), Molecular Devices (Sunnyvale, CA), Scripps (San
Diego,
CA), Corning (Kennebunck, MA), and Whatman (Clifton, Nn.
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Preparation of TG and BSA-Glyphosate
Conjugates of glyphosate with TG or with BSA were prepared by activating
the carboxylic groups of TG or BSA with 1-ethyl-3-(3-diaminopropyl)
carbodiimide
hydrochloride (EDC) followed by their coupling to the secondary amino group of
5 glyphosate as follows. Fifty mg of EDC and S mg of sulfo-NHS
(Sulfo-N-hydroxysuccinimide) were added to 15 mg of TG pre-dissolved in 5 ml
of
10 mM KHZP04, pH 5Ø The reaction mixture was stirred for 2-3 minutes
followed
by the addition of 5 ml of a 2% solution of glyphosate in 0.2 M KZHP04, pH
8.5.
After the solution was stirred overnight, the conjugate (TG-glyphosate) was
dialyzed
10 exhaustively against PBS-7.4 (0.14 M NaCI, 10 mM K2HP04, pH 7.4) or TBS-8
(tris-buffered saline, pH 8). The BSA-glyphosate conjugate was prepared using
the
same procedure. Both conjugates were stored at s-15°C. By this
procedure the
conjugate was formed by linkage to the carrier protein predominantly via the
glutamic
and aspartic acid residues.
15 EXAMPLE 2
Preparation of TGGlyphosine
A conjugate of TG and glyphosine was prepared by activating the carboxylic
group of glyphosine with EDC followed by its coupling to the amino groups of
TG as
follows. One hundred mg of EDC and 10 mg of Sulfo-NHS were added to 465 mg of
20 glyphosine pre-dissolved in 2.5 ml of 10 mM KH2P04, pH 5Ø After the
reaction
mixture was stirred for 2-3 minutes, the entire solution was transferred to a
second
reaction vessel containing 15 mg of TG pre-dissolved in S ml of 0.2 M KZHP04,
pH 8.5. The final reaction mixture was stirred overnight, followed by
exhaustive
dialysis against PBS at a pH of 7.4 or TBS at a pH of 8. The conjugate was
stored at
s -15°C. By this procedure the conjugate was formed by linkage to the
Garner
protein predominantly via the epsilon-amino groups of lysine residues.
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EXAMPLE 3
Production of Antibodies
Antibodies to TG-glyphosate conjugate and TG-glyphosine conjugate were
produced in New Zealand white rabbits as follows. Rabbits were immunized with
0.5-1.0 mg of immunizing conjugate per rabbit per injection. The immunizing
conjugate was emulsified with Complete Freund's Adjuvant for primary
injections
and with Incomplete Freund's Adjuvant for booster injections. Three or four
booster
injections were performed at monthly intervals to raise the desired titer to
10 to SOK.
The rabbits were bled on 1213 days following each booster injection. Antisera
were
monitored for titer and analyte specificity by capturing the relevant
antibodies on
ELISA plates coated with BSA-glyphosate conjugate. The captured antibodies
were
measured in a subsequent step by incubating the plates with an excess of goat
anti-rabbit-IgG-horseradish peroxidase (GARIG-HRP).
EXAMPLE 4
Preparation of HRP-Glyphosate
An oligomeric form of HRP-glyphosate was prepared as follows. Thirty mg
of HRP, pre-dissolved in 7.5 ml Buffer A (0.1 M sodium acetate, 0.15 M sodium
chloride, pH 5.5), was stirred with 40 mg sodium m-periodate (25 mM) for 40
minutes in an ice-bath, followed by quenching with 0.3 ml of ethylene glycol
for 5
minutes. The reaction mixture was dialyzed for approximately 4 hours against
two
liters of 1:3 diluted Buffer A and the dialysate was then mixed with 2 ml of
0.5 M
adipic acid dihydrazide in Buffer A. The reaction mixture was stirred at room
temperature for about 1 hour and then at 2-6°C overnight, followed by
extensive
dialysis at 2-6°C against 1:3 diluted Buffer A. HRP-hydrazide prepared
above was
stored at 2-6°C. Next, a second portion of HRP was coupled to
glyphosate as follows.
Twenty mg EDC and 2 mg sulfo-NHS were added to 5 mg HRI' (pre-dissolved in 2
ml of 10 mM KH2P04, pH 5.0) and the reaction mixture was stirred for 2-3
minutes
followed by the addition of 2 ml of a 2% solution of glyphosate in 0.2 M
K2HP04, pH
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8,5. After stirring the reaction mixture for 1 hour at room temperature and
then
overnight at 2-6°C, the conjugate (HIU'-glyphosate) was dialyzed
exhaustively at
2-6°C against Buffer A. Finally, the I-iRP-glyphosate conjugate (5 mg)
was further
coupled to multiple copies of HRP-hydrazide (30 mg) as follows. HRP-glyphosate
(S
mg in 5 ml Buffer A) was oxidized with 25 mM sodium m-periodate using
essentially
the same procedure as described above for the preparation of HItP-hydrazide.
The
dialysate from this reaction was combined with I-iRP-hydrazide prepared
previously
and the reaction mixture was stirred at room temperature for 30 minutes and
then
overnight at 2-6°C. The final conjugate was stored at 5 -15°C in
the presence of 50
mM Trizma-8 (Sigma), 1% BSA, 0.01% thimerosal and 50% glycerol. The working
aliquots of this conjugate can be stored at 2-6°C for at least 6
months.
EXAMPLE 5
Linker-Assisted ELISA for Glyphosate Using Antigen Coated Plates
On the basis of theoretical considerations, the antibodies raised against the
TG-glyphosate and TG-giyphosine conjugates (i.e., the anti-TG-glyphosate and
anti-
TG-glyphosine antibodies) were expected to exhibit low affinity toward unbound
glyphosate, a low molecular weight.compound devoid of any rigid ring
structure. In
one ELISA format featuring antigen-coated microtiter plates, solid phase BSA-
glyphosate competes against unbound glyphosate in the assay mixture, for
binding to
a limited amount of antibody in the assay mixture. In such assays, unbound
glyphosate at concentrations lower than 2000 ng/ml is not able to compete
effectively
with the solid phase BSA-glyphosate, resulting in a relatively insensitive
assay (See
Figure 1). The antibody, however, shows high af~mity toward the coating
conjugate
BSA-glyphosate, since the latter resembles the immunizing conjugates.
The present experiment was conducted to determine whether the affinity of the
anti-TG-glyphosate and anti-TG-glyphosine antibodies prepared in Example 3
above
could be enhanced by chemically modifying the analyte glyphosate in the sample
to
resemble the relevant epitopic structure of the immunogen and plate antigens
used in
a competition ELISA. Since the TG-glyphosate and TG-glyphosine conjugates were
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23
formed by linkage of glyphosate or glyphosine to the carrier proteins via
amide or
imide bonds involving glutamic acid, aspartic acid, and lysine residues of TG,
it was
contemplated that the affinity of the anti-TG-glyphosate and anti-TG-
glyphosine
antibodies for the analyte glyphosate in a sample could be enhanced by
derivatization
of analyte glyphosate with aspartic and glutamic acids, thereby incorporating
an imide
linkage into the analyte to resemble the relevant epitopic structure found on
the
immunogen and the plate antigen.
Initially, a mixture of aspartic and glutamic acids was tested for efficacy in
affinity enhancement. The linkers, aspartic and glutamic acid, were activated
with
EDC for 2-5 minutes and then allowed to couple to glyphosate for about 30 min.
via
the secondary amine function of the glyphosate. A series of glyphosate-linker
compounds were prepared by the same procedure using succinic, glutaric,
adipic,
N-acetyl-aspartic, N-acetyl glutamic, poly-aspartic, and poly-glutamic acids,
and
succinic and glutaric anhydrides as linkers. The anhydrides do not require pre-
activation with a compound such as EDC.
It was discovered that the conjugation of glyphosate with the linker molecules
resulted in a marked improvement in assay sensitivity. Highest enhancement in
assay
sensitivity was achieved with glutaric and succinic anhydrides.
The effect of pH on the assay was also investigated. As expected, the
antibodies of the present invention bound derivatized glyphosate much more
tightly at
pH 9 than at pH 7. The binding was almost completely abolished at pH 5, most
likely
due to diminishing negative charge on the acidic groups of glyphosate.
Furthermore,
these antibodies exhibited virtually no cross-reactivity (less than 0.01%)
toward
aminomethylphosphonic acid (a major metabolite of glyphosate), thereby
suggesting a
critical role of the negative charges in antibody binding.
Based upon the foregoing, one embodiment of the invention, featuring
antigen-coated plates, comprises the following steps:
1. Microtiter plates (Costar High Binding) are coated with BSA-
glyphosate (Osborn reagent No. R0788), 0.2 ml/well, at 14 nglml water. After a
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24
coating period of 16-24 hours, the plates are over-coated with 1% BSA, 0.21
ml/well.
Finally, the plates are rinsed twice with water, air-dried overnight and then
stored at
2-6°C for up to at least two months.
2. 1 Sx75 mm assay tubes are labeled with appropriate ID numbers and
10 p,l of 0.5 M NaHC03 dispensed per tube.
3. 100 ~tl of each sample (standards, controls and unlrnowns) is dispensed
per assay tube.
4. 20 microliters of a 5% solution of glutaric anhydride in DMSO are
added per assay tube. Vortex and incubate the tubes at room temperature for
approximately 30 minutes.
5. Anti-TG-glyphosine (Osborn reagent No. 80881) is diluted 1:50,000 in
IB-9 (50 mM Trizama-9~, 100 mM NaCI, 1% BSA, 0.1% Tween-20, 0.01%
T'himerosal, 2.5 ppm Bromcresol Purple) and 700 pl of this solution is
dispensed into
each of the assay tubes. Vortex the tubes and then incubate them on a shaker
for 10-
20 minutes.
6. Each sample is loaded into triplicate wells of a pre-coated plate (see
step 1) and the plate is incubated on a shaker for approximately 1 hour.
7. The plate is washed once with EWB (0.85% NaCI, 0.005% Triton X-
100) using an automatic plate washer, GARIG-HRP (1:1000 dilution of Osborn
reagent No. 80843 in IB-9) is added, and the plate is incubated on a shaker
for
approximately 45 minutes.
8. Finally, the plate is washed three times with EWB, and a
tetramethylbenzidine-based HRP substrate is added (200 pl/well). The plate is
incubated on a shaker for 10-20 minutes followed by the addition of 100 pl of
Stop
Solution/well (1N HCl). The plate is read at 450 nm using a computer-
interfaced
ELISA reader (V-max, Molecular Devices). Glyphosate concentration in the
unknown samples is estimated from a concurrently-run standard curve.
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EXAMPLE 6
Linker-Assisted ELISA for Glyphosate Using Antibody-Coated Plates
Preparation of Pre-coated Plates
Antibody coated plates were prepared in batch using the following procedure.
5 Microtiter plates (Costar High Binding) were coated overnight with protein A
{2
p,g/ml, 0.19 ml/well) in 0.2 M sodium bicarbonate. The plates were washed once
with
ELISA wash buffer (0.85% NaCI, O.OOOS% Triton X-100). Rabbit anti-TG-
glyphosate was diluted (e.g. 1:10,000, depending on the titer) with antibody
incubation buffer (50 mM Trizma 9.1, 100 mM NaCI, 1% BSA, 0.1% Tween 20,
10 0.1% sodium azide), followed by dispensing of this solution (0.2 ml/well)
into the
wells of protein A coated plates. The plates were again incubated overnight
and then
washed twice with ELISA wash buffer containing 5% sucrose. Finally, the plates
were air-dried overnight, sealed with plate sealing film, and stored at 2-
6°C for up to
at least three months. Finally, the plates were rinsed twice with deionized
water,
15 air-dried overnight and then stored at 2-6°C for up to at least two
months.
Preparation of Buffer-coated Assay Tubes
A batch of assay tubes (15x75 mm polypropylene) were prepared by
dispensing 20 pl of 0.5 M Trizma 9.1 (Sigma Chemicals) per tube and allowing
complete drying (1-2 days at room temperature). These ready-to-use assay tubes
can
20 be stored at room temperature for at least 6 months.
Preparation of Succinylation Reagent
A stock solution of 2% succinic anhydride in dimethylsulfoxide (DMSO) was
aliquoted into 2-ml portions. This reagent can be stored at 2-6°C for
up to at least 3
months.
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The ELISA method
The presently preferred ELISA method of the present invention was carried
out as follows: Buffer-coated assay tubes were labeled with appropriate ID
numbers
and 0.2 ml of each sample (standards, controls and unlaiowns) was dispensed
per
assay tube. To each tube was added 25 p.l of succinylation reagent and the
tubes were
vortexed and then incubated at room temperature for approximately 20 minutes.
Then
the stock solution of HRP-glyphosate conjugate (prepared as in Example 4) was
diluted 1:100 in IB-0.2 solution (0.2 M Trizma~ 9.1, 1% BSA, 0.1% Tween~-20,
0.02T Thimerosal) and 0.6 ml of the dilute solution was added per assay tube.
Each sample was vortexed and loaded into triplicate wells (0.2 ml/well) of the
pre-coated plate. The plate was sealed and incubated on a shaker for
approximately
40 minutes, then washed three times with ELISA wash buffer, then 200 pl/well
of a
pre-formulated tetramethylbenzidine-hased HRP substrate was added. The plate
was
incubated on a shaker for approximately 10 minutes, the reaction was stopped
by
adding 100 pl/well of stop solution (1N HCl), and the plate was read at 450 nm
using
a computer interfaced ELISA reader (Molecular Devices, Sunnyvale, CA).
Glyphosate concentration in the unknown samples was estimated by comparison
with
a concurrently-run standard curve.
EXAMPLE 7
Glyphosate SPE Procedure
Solid phase extraction (SPE) for matrix cleanup or for concentration of
samples
was performed according to the following procedure.
Sample tubes (12x75 mm polypropylene tubes precoated with 20 E.tM Tris base),
elution tubes (untreated 12 X 75 mm polypropylene tubes), and SPE columns
(VVhatman~ SPE columns, SAX,1 ml, 100 mg) were labeled with appropriate ID
numbers, and the columns were preconditioned with 1 ml deionized water (di
H20),
using positive pressure to move the liquid through the column.
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Samples (standards, quality controls, and unlalowns) were prepared by adding 2
ml of sample to sample tubes and vortexing. A 1 ml aliquot of each sample was
loaded
onto the appropriate preconditioned column, using positive pressure to move
the liquid
through the column at a flow rate of approximately 1 ml/minute. The procedure
was
repeated using the remaining 1 ml of sample. Each column was then washed with
1 ml
di H20, columns were transferred to appropriately labeled elution tubes,
samples were
eluted with 1 ml Eluent Solution (0.2 N HCl) using positive pressure to move
the liquid
through the column at a flow rate of approximately 1 ml/minute, and tubes were
vortexed.
The eluates were saved for glyphosate analysis by ELISA (Example 6).
While the invention has been described in detail with reference to certain
preferred embodiments thereof, it will be understood that modifications and
variations
are within the spirit and scope of that which is described and claimed,
