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Food and Agricultural Immunology
ISSN: 0954-0105 (Print) 1465-3443 (Online) Journal homepage: http://www.tandfonline.com/loi/cfai20
Enzyme-linked immunoassay for the detection ofglyphosate in food samples using avian antibodies
A. Arul Selvi , M.A. Sreenivasa & H.K. Manonmani
To cite this article: A. Arul Selvi , M.A. Sreenivasa & H.K. Manonmani (2011) Enzyme-linkedimmunoassay for the detection of glyphosate in food samples using avian antibodies, Foodand Agricultural Immunology, 22:3, 217-228, DOI: 10.1080/09540105.2011.553799
To link to this article: http://dx.doi.org/10.1080/09540105.2011.553799
Published online: 16 Mar 2011.
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Enzyme-linked immunoassay for the detection of glyphosate in foodsamples using avian antibodies
A. Arul Selvia, M.A. Sreenivasab and H.K. Manonmania*
aFermentation Technology and Bioengineering Department, Central Food TechnologicalResearch Institute, Mysore 570 020, Karnataka, India; bFood Safety and Analytical QualityControl Laboratory, Central Food Technological Research Institute, Mysore 570 020,Karnataka, India
(Received 1 July 2010; final version received 8 January 2011)
A simple competitive immunoassay was developed for the measurement ofglyphosate in food samples. It employed the avian antibodies (IgY) thatrecognised glyphosate as a capture reagent and glyphosate�alkaline phosphataseconjugate as an enzyme label. The assay depended on the competitive bindingbetween the antibody and glyphosate derived from food samples for binding siteswith immobilised glyphosate�OVA conjugate. The concentration of glyphosate inthe food samples was quantified by the ability of the pesticide present in foodsamples to inhibit the binding of the enzyme conjugate to the antibody andsubsequently the colour formation in the assay. The assay was specific toglyphosate with a limit of detection of 2 ppb. Mean analytical recovery ofglyphosate in different food samples was 9.00�134.00%. The precision of theassay was satisfactory. The assay compared favourably with high-pressure liquidchromatography (HPLC) in its ability to accurately measure glyphosate in thefood samples.
Keywords: glyphosate; avian antibodies (IgY); enzyme-linked immunosorbentassays; herbicide; food samples; high-pressure liquid chromatography; immunol-ogy; hapten
Introduction
Glyphosate [N-(phosphonomethyl) glycine] is a broad-spectrum, non-selective,
systemic, post-emergence herbicide used both agriculturally and domestically
(Mensink & Janssen, 1994). It is an active ingredient in a number of commercial
herbicides produced by Monsanto, Cheminova and Zeneca Corp. Various formula-
tions are scheduled for use that include products with the trade names Clear It,
Expedite, Ezject, Glyfos, Laredo, Renegade, Roundup, Touchdown 480, Vision and
Wrangler. Glyphosate is a member of the amino acid herbicide family, and its mode
of action is through inhibition of 5-enolpyruvylshikimate-3-phosphate synthase
(EPSP), an enzyme of the shikimic acid pathway. This enzyme is important in the
biosynthesis of the aromatic amino acids � phenylalanine, tyrosine and tryptophan.
A blockage of the shikimic acid pathway leads to a depletion of the free pool of
aromatic amino acids in higher plants (Duke, 1988). Glyphosate translocates readily
in the plant, making it effective for controlling perennial weeds and overwintering
*Corresponding author. Email: [email protected]
Food and Agricultural Immunology
Vol. 22, No. 3, September 2011, 217�228
ISSN 0954-0105 print/ISSN 1465-3443 online
# 2011 Taylor & Francis
DOI: 10.1080/09540105.2011.553799
http://www.informaworld.com
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rhizomes and tubers. It is registered for preplant or postharvest treatment in crops
and on non-crop land (Omafra staff, 2002). The continued use of glyphosate
indiscriminately by agriculturalists raises the potential for residue accumulation in
food and water commodities. The maximum residue limit (MRL) concentration for
food crops has been set at 1.0 mg/ml by the Indian Prevention of Food Adulteration
act (PFA), the Canadian Food and Drug Act as well as the US Food and Drug
Regulations (USFDA). Glyphosate is persistent in soil with a half-life of 47 days(Wauchope, Buttler, Hornsby, Augustijn Beckers, & Burt, 1992). In many countries,
toxicology studies have been conducted to enable evaluation of the potential health
risk (Williams, Kroes, & Munro, 2000). Glyphosate’s laboratory testing has
discovered some negative short-term and long-term health effects. Additionally,
the glyphosate-containing product Roundup has been known to be used in suicide
cases in Japan and consumption of it results in symptoms such as intestinal pain,
vomiting, excess fluid in the lungs, pneumonia, clouding of consciousness and
destruction of red blood cells. Short-term exposure to glyphosate can cause breathing
difficulties, loss of muscle control and convulsions. Some glyphosate-containing
products belong to Toxicity Category I and II (acutely toxic) and are toxic to animals
and humans, and some symptoms include eye and skin irritation, headache, nausea,
numbness, elevated blood pressure and heart palpitations (Cox, 2000). Although
glyphosate is an acid, it is commonly used as the isopropylamine or trimethylsulfo-
nium salts and is usually distributed as water-soluble concentrates and powders.
Glyphosate analysis in environmental matrixes is problematic because it is a smallmolecule and has structural similarity to many naturally occurring plant materials
such as amino acids and secondary plant compounds. It is highly soluble in water
thereby making its extraction with solvents difficult. Therefore, glyphosate isolation
and quantitation poses a challenge to the analytical chemist due to the necessity of
removing matrix effects before analysis. In the last few years, many techniques have
been tested for analysis of glyphosate, including ion chromatography with UV
detection by Bedry and Parrot (1998), fluorescence detection (Mallat & Barcelo,
1998), conductivity detection (Zhu, Zhang, Tong, & Liu, 1999) or mass spectro-
metric detection (Bauer, Knepper, Maes, Schatz, & Voihsel, 1999). These methods
are not, however, sufficiently sensitive to reach the quality threshold for drinking
water. Gas chromatography continues to be of interest, although it suffers from
problems such as tedious sample preparation to clean-up and convert the molecules
into volatile derivatives (Borjesson & Torstensson, 2000). Another technique
investigated is high-pressure liquid chromatography (HPLC) with post-column
derivatisation with o-phthaldealdehyde-mercaptoethanol and fluorescence detection
(Reupert & Schlett, 1997; Winfield, 1990). The most difficult part of the analysis is,
in fact, the extraction of the compounds from food materials, so derivatisation ofglyphosate is usually conducted in this medium. The detection achieved using these
methods is generally at higher concentrations than those typically obtained using
enzyme-linked immunosorbent assays (ELISA) for most herbicides (Rubio et al.,
2003).
ELISA is therefore said to be a valuable tool in residue analysis and complements
conventional analytical methods (Johnson & Hall, 1996; Parnell & Hall, 1998).
ELISA provides rapid sample testing and accurate results and is more cost-effective
than conventional chromatographic analysis (Hall, Deschamps, & McDermott,
1990). ELISA has been used successfully for the quantitative analysis of numerous
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pesticides in food matrices with little or no matrix interference (Lawruk et al., 1994;
Rubio, Itak, Scutellaro, Selisker, & Herzog, 1991).Previous publication on the indirect immunosorbent assay for the detection and
quantitation of glyphosate described by Clegg, Stephenson, and Hall (1999) using
mammalian antibodies (IgG) showed the limit of detection of 0.076 mg mL�1 which
indicates less sensitivity in comparison with the present studies which showed the
detection limit of 2 ng mL�1 using avian antibodies (IgY). This implies that the hen’s
antibodies (IgY) in particular have clear advantages over mammalian immunoglo-
bulins (Schade, Behn, Erhard, Hlinak, & Staak, 2001).
In this paper, we report the quantitative performance of ELISA for glyphosate
detection and quantitation in environmental food samples using avian antibodies.
Material and methods
Chemicals and reagents
Bovine serum albumin (BSA), Freund’s complete adjuvant, Freund’s incomplete
adjuvant, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Ovalbumin (OVA),
2,4,6-Trinitrobenzenesulfonic acid (TNBS) and other pesticides standards were
purchased from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA). The
analytical standard of glyphosate (99%) was obtained from Monsanto Co (St. Louis,
MO, USA). Rabbit anti-chicken IgY conjugated to alkaline phosphatase (ALP), was
purchased from Bangalore Genei (Bangalore, India). Microtiter plates were
purchased from NUNC (Roskilde, Denmark). ELISA plates were analysed using a
VERSAmax tunable microplate reader (Silicon valley, CA, USA). Dialysis cassettes
were purchased from Thermo Scientific (Rockford, IL, USA). All the other
chemicals used were of analytical grade and purchased from standard chemical
companies. Food samples such as turmeric, chili, tea samples from different regions,
coffee, coriander, ginger and rice were purchased from local market.
The hapten, OVA-pesticide conjugate and anti-glyphosate antibody for glypho-
sate assay were produced at CFTRI, Mysore, using 22-week-old single comb white
leg horn poultry that were purchased from Kateel Poultry Farm (Mysore, India).
Synthesis of pesticide�protein�hapten conjugates
The synthetic route for the preparation of conjugate was devised. The glyphosate�BSA immunogen was prepared by dissolving the acid form of glyphosate (0.3 mM)
and 100 mg of BSA in 10.0 mL of 0.1 M phosphate-buffered saline (PBS). This
solution was mixed with 130 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
and allowed to react for 2 h at room temperature on a magnetic stirrer. The solution
containing the glyphosate�protein (gly-BSA) conjugates was dialysed extensively
using the dialysing cassettes (10K MW cutoff) against distilled water for 24 h at 48C.
The extent of conjugation was determined by using TNBS method (Plapp, Moore, &
Stein, 1971). Coating conjugate of glyphosate (gly-OVA) was synthesised by
conjugating glyphosate to ovalbumin (OVA). The conjugates thus obtained were
lyophilised and stored at �208C till further use.
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Estimation of the level of conjugation
The TNBS reaction was carried out as follows. 200 mL of 0.6 M sodium borate
buffer, pH 9.5, was mixed with 200 mL of a solution of conjugate in 0.05 M sodium
acetate buffer, pH 4.7 and 50 mL of 0.2 M NaOH. The reaction was initiated by the
addition of 50 ml of a freshly prepared solution of TNBS (7.2 mg mL�1 of water). The
reaction mixtures were kept at 258C in dark for 60 min and then the absorbance at
367 nm was measured.
Immunisation of poultry
Twenty-two-week old poultry were injected intramuscularly, with 1 mg of immuno-
gen (Gly-BSA conjugate) in 0.5 mL of a 50 mM PBS, pH 7.5 and 0.5 mL Freund’s
complete adjuvant. The secondary immunisations were repeated once in 15 days for 6
months with 1 mg of immunogen in 0.5 mL of a 50 mM PBS, pH 7.5 and 0.5 mLFreund’s incomplete adjuvant. The eggs were collected everyday for the isolation of
antibodies.
Anti-glyphosate antibody isolation
Yolk was separated from egg white and suspended in PBS. The egg yolk was brokenand made into uniform suspension by mixing well in a magnetic stirrer for 10 min.
Mixing was continued for 30 more minutes with the addition of chloroform at room
temperature. The mixture was centrifuged at 10,000 g, 48C for 10 min. The
supernatant was decanted and Polyethylene Glycol 6000 was added at 14% (W/V)
level. Mixing was continued for 30 min at room temperature. Antibody was
separated by centrifuging at 10,000 g, 48C for 10 min. The precipitate (IgY) obtained
was dissolved in required quantity of PBS and stored at �208C (Schade et al., 2001).
Immunoassay procedure
Glyphosate-specific antibody titres were monitored as described by Campbell,
Burdon, and Knippenberg (1984) and Gee, Miyamoto, Goodrow, Buster, and
Hammock (1988). The protein concentration of IgY was determined by Bradford
method (1976). The optimum antibody concentration for coating onto the microwellplates and the best working concentration of the enzyme conjugate were determined
by checkerboard titration. The coating conjugate (glyphosate�OVA conjugate) was
diluted in carbonate buffer (50 mM, pH 9.6) at concentrations of 3.125 mg mL�1
through 625 pg mL�1/ml and added to the wells of the microwell plates (100 mL).
The plates were incubated at 48C overnight and were washed with 0.1% Tween 20 in
PBS (137 mM NaCl, 3 mM KCl and 10 mM sodium phosphate, pH 7.4) (PBS-T),
and the wells were blocked with 0.2% gelatine for 1 h. After a wash with PBS-T, anti-
glyphosate antibody (IgY) diluted in carbonate buffer at concentrations of 34.9 mgmL�1 through 69.5 ng mL�1 was coated onto microwell plates (100 mL). The plates
were incubated for 1h at 378C. After a thorough wash in PBS-T, anti-chicken rabbit
antibody (1: 10,000) conjugated with ALP was added (100 mL). After 1 h at 378C, the
plates were washed well in PBS-T. Colour was developed using p-nitrophenyl
phosphate (pNPP) (1 mg mL�1) in 1% diethanolamine buffer, pH 9.8 (150 mL). The
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reaction was stopped by the addition of 50 mL of 3 M NaOH. The absorbance was
measured in an ELISA reader at 405 nm. Concentrations of antibody and enzyme
conjugate that yielded a signal between 0.8 and 1.2 absorbance units were used for
further testing of the competitive immunoassay procedures.
Optimisation of glyphosate concentration by ELISA
Coating conjugate was loaded to microwell titre plates (100 mL/well) and allowed to
incubate overnight at 48C. The plates were washed well with PBS-T and patted dry on
paper towels. Sites not containing coating conjugate were blocked with 100 mL/wellof 0.2% gelatin. After 60 min incubation, the plates were washed and dried as
previously described. Glyphosate standards or samples containing glyphosate were
prepared in carbonate buffer. Anti-glyphosate antibody and sample or standard
solutions were mixed 1:1 (v/v) and allowed to incubate in test tubes for 60 min. The
pre-incubated mixtures were transferred to the plates (100 mL/well). The plates were
incubated for another 60 min at room temperature before being washed with PBS-T.
The secondary antibody tagged with ALP was added to each well (100 mL) and
incubated at 378C for 60 min. The wells were then washed as described earlier. About150 mL of pNPP in diethanolamine buffer was added and incubated for 30 min.
Optical density (OD) at 405 nm was recorded after stopping the reaction with 50 mL
of 3 M NaOH.
Standard curve
The absorbance at 405 nm is inversely proportional to the concentration of
glyphosate in the standards and samples. To normalise the absorbance, the
background absorbance was subtracted from each value followed by division of
each value by the positive control (0 ng ml�1 glyphosate). The standard curves were
constructed by plotting the normalised absorbance values (A/A0) against the log
values of the glyphosate concentration. Glyphosate concentrations in the food
samples were interpolated from the standard curve.
Cross-reactivity
Few pesticides were tested for cross-reactivity to the anti-glyphosate antibody.
Glyphosine [N, N-bis (phosphonomethyl) glycine], a related herbicide, and few
structurally related smaller molecules were tested for their cross-reactivity. A 1000
mg mL�1 standard of each test chemicals was prepared using the carbonate bufferand tested against the anti-glyphosate antibodies. The IC50 value, per cent cross-
reactivity and least detectable dose for each of the compounds were determined.
High-pressure liquid chromatography (HPLC)analysis
Sample preparation was carried out by weighing one gram of food sample into a50 mL polypropylene centrifuge tube with stopper. About 10 mL of ultrapure
water and 5 ml dichloromethane were added. The tube was sonicated for 10 min
and shaken thoroughly for 1 min that was followed again by 10 min of sonication.
After that the tube was centrifuged for 10 min at 8000 g. The upper water phase,
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approximately 9 mL, was transferred to another polypropylene tube. Evaporation
to dryness with stream of N2 at 558C on water bath was followed, and the sample
was redissolved in 1 mL of carbonate buffer prepared using ultrapure distilled
water.
Glyphosate was detected by HPLC using a cation exchange analytical column.
The compound was hydrolysed at 368C (Clegg & Ripley, 1996) with sodium
hypochlorite to form glycine. The glycine was then reacted with o-phthalaldehyde
(OPA) in the presence of mercaptoethanol at 558C to produce a highly fluorescent
isoindole, which was detected fluorometrically (excitation 330 nm, emission 465
nm). All glyphosate standards were prepared in distilled water and serially diluted
in potassium dihydrogen phosphate buffer to the concentration range required for
analysis. A volume of 10 mL of standard/sample was injected into the liquid
chromatograph, which had a mobile phase of potassium dihydrogen phosphate
buffer (0.005 M) with a flow rate set at 1.0 ml/min. The total chromatographic run
was 30 min. The mobile phase of potassium dihydrogen phosphate buffer was
isocratic for 17 min, column regenerant (potassium hydroxide) for 2 min and
finishing the chromatographic run with an 11 min continuation of potassium
dihydrogen phosphate buffer mobile phase. Peak areas of the standards were
plotted against the concentration of glyphosate, and the resulting standard curve
was used to interpolate glyphosate concentrations in the food samples.
Results and discussion
This study describes a sensitive enzyme immunoassay that quantifies glyphosate in
food samples using avian antibodies. The food sample containing glyphosate
competes with anti-glyphosate IgY which will be recognised by immobilised
glyphosate�OVA antigen (coating conjugate) in microwell plates. After removal of
unbound reagents, the amount of antibody bound to the immobilised glyphosate�OVA conjugate was determined by using a chromogenic substrate. The concentra-
tion of glyphosate in a food sample was quantified by the ability of the glyphosate
in food material to inhibit binding of anti-glyphosate antibody to the conjugate,
and the colour development was inversely proportional to the concentration of
glyphosate in the original sample.
It was observed that with the glyphosate�BSA conjugate, out of total 28 available
lysine molecules in BSA, 19 lysine molecules were found to be bound to glyphosate
molecule, i.e. the extent of conjugation was 68%.
Optimum assay conditions
The optimum concentrations of anti-glyphosate antibody required and the best
working concentration of the conjugate required for coating on to the microwell
plates were determined by checkerboard analysis. The analysis indicated that
antigen (glyphosate�OVA conjugate) dilution of 1:2.5 lakhs with carbonate buffer
(125 pg protein) and antibody dilution of 1:25 K (139 ng protein) gave the
optimum readings (data not shown). These concentrations were chosen for further
work.
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Stability of anti-glyphosate antibody
Antibody stored at 48C and �208C was checked at different intervals of time for theamount of antibody activity. As shown in Figure 1, the antibody could be stored for
nearly six months at 48C. The loss in activity was 37% by the end of six months of
storage period at 48C. Thus the antibody (IgY) could be stored at 48C with little
activity loss up to six months. This reduces the cost of antibody production if stored
properly. The loss in activity for antibody stored at �208C was �2% by the end of six
months. Chicken egg yolk antibodies have been shown to have good activity up to 12
years (Schade et al., 2001) at �408C. It is clear that avian antibodies have longer half-
life than mammalian antibodies (IgG). These antibodies would be better choice tomammalian counterparts (Schade et al., 2001).
Calibration curves and sensitivity
Calibration curve was generated using glyphosate (99% pure) at concentrations from 2
to 1000 ng mL�1, prepared in carbonate buffer (Figure 2). The sensitivity of the assaywas determined by identifying the limit of detection defined as the lowest measurable
concentration of glyphosate that could be distinguishable from zero concentration. On
the basis of eight replicate measurements, the limit of detection was 2 ng mL�1. The
IC50 value of this avian antibody was 36.30 ng mL�1. In the similar studies by Clegg et
al. (1999) using IgG, the limit of detection was 0.076 mg mL�1 with an IC50 value of
1.54 mg mL�1 with anti-glyphosate rabbit antibody showing slightly less sensitivity.
Analytical performance of the assay
The glyphosate at different concentrations (2�250 ng mL�1) spiked to different food
samples was extracted using ultrapure water, and immunoassay was carried as
00
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4 5 6Storage period (months)
O.D
. 405
4°C
–20°C
Figure 1. Stability of anti-glyphosate antibody.
Notes: Antibody stored at 48C and �208C was checked at different intervals of time for the
amount of residual antibody activity. Data points represent the mean of triplicate
determinations. The standard deviation in the mean is depicted by the error bars.
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described in the experimental section. Each sample was analysed in triplicate for its
glyphosate content. The limit of detection of glyphosate in different food matrices
varied (Figure 3). The background effect of the glyphosate extract from turmeric, tea
and rice was very less. With chili and coffee as substrates for spiking, interference
could be observed. Ginger had tremendous interference with immunoassay. Linear
regression analysis of the results yielded a linear equation: y�0.9681x�0.3409,
R2 �0.9998 for turmeric, y�0.6618x�3.2148, R2 �0.9954 for chili, y�0.9792x�0.6683, R2 �0.9997 for tea (Assam), y�0.9825x�0.1669, R2 �0.9996 for coriander,
y�0.9638x�0.6991, R2 �0.9996 for coffee, y�0.9816x�0.1925, R2 �0.9998 for tea
(Ooty), y�0.9725x�0.6264, R2 �1 for rice and y�0.6733x�1.734, R2 �0.9895 for
ginger.
20
00 500 1000
40
60
80
100
120
y = 10.059x – 14.094R2 = 0.09649
Glyphosate concentration (ng mL–1)
Inh
ibit
ion
(%
)
Figure 2. Immunoassay with different concentrations of glyphosate.
Notes: The concentrations of the pesticide standard quoted on the X-axis (ng mL�1) were
diluted using carbonate buffer. Data were mean of eight independent experiments (n�8).
350
300
250
200
150
100
50
0
Turmeric Chili
Tea (Assam)
Tea (ooty)
RiceGinger
CorianderCoffee
Food materials
Co
nce
ntr
atio
n o
f g
lyp
ho
sate
(n
g)
2ng10ng100ng250ng
Figure 3. Immunoassay of glyphosate spiked to different food samples.
Notes: Data points represent the mean of three independent experiments (n�3). The standard
deviation in the mean is depicted by the error bars.
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Specificity of the assay
The specificity of the assay for glyphosate was studied. The presence of other
pesticides at concentrations of 1000 ng mL�1 level was tested. Figure 4 shows the
cross-reactivity of different individual pesticides added in the assay. These data were
based upon triplicate determinations. The presence of any of the herbicide did not
interfere in the assay, i.e. the cross-reactivity was very low and it ranged between 1
and 5%. Glyphosine, a structurally related herbicide cross-reacted by 22% with anti-glyphosate antibody.
Comparison of immunoassay with high-pressure liquid chromatography (HPLC)
The mean analytical recovery was calculated as the ratio between the glyphosate
concentration found and the concentration added, and expressed as percentage.
Glyphosate-spiked food samples at 2�250 ng mL�1 levels were checked by both
immunoassay and HPLC as described in the experimental section. The comparisons
of these results are shown in Table 1. The recovery of glyphosate by immunoassay
(Table 1a) with turmeric as matrix was 85 and 95%, respectively, at 2 and 250 ngmL�1. Chili showed 88 and 60% recovery at 2 and 250 ng mL�1 level. This may be
due to the matrix effect of the sample. Glyphosate in tea could be recovered at 74 and
82%, respectively, at 2 and 250 ng mL�1. Glyphosate spiked to coriander could be
recovered by 53 and 80%, respectively, at 2 and 250 ng mL�1. Coffee-spiked
glyphosate had very low recovery of 9 and 56%, respectively, at 2 and 250 ng mL�1.
About 71 and 96% of the added glyphosate could be recovered from rice-spiked
samples. Ginger had the lowest recovery of 11 and 5%, respectively, at 2 and 250 ng
mL�1. This may be due to the interference of the matrix effect. The recovery ofglyphosate by HPLC (Table 1b) in turmeric was 91 and 96% at 2 and 250 ng mL�1
levels, respectively. In chili, it was 61 and 66%, respectively, for 2 and 250 ng mL�1.
In tea, coriander, coffee and rice, the recovery was almost complete. In ginger, only
52 and 66% of the added glyphosate could be recovered. The immunoassay method
1009080706050
Inh
ibit
ion
(%
)403020100
Glypho
sate
Atrazin
e de
seth
yl
Mon
ocro
toph
os2,
4-D
Parat
hion
Met
hyl P
arat
hion
Aceph
ate
Glypho
sine
Dichlor
ovos
Atrazin
e
Simaz
ine
Terbu
tryn
Diffrent pesticides used
Figure 4. Cross-reactivity/specificity of glyphosate immunoassay.
Notes: Few pesticides were tested for cross-reactivity to the anti-glyphosate antibody. Data
were based upon the mean of triplicate determinations. Competitive immunoassays were
performed as described in the experimental section using analytical grade standard pesticides.
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compared well with HPLC, with few food materials indicating the ability of
immunoassay to accurately determine glyphosate in certain food samples.
Conclusion
A sensitive immunoassay for the determination of glyphosate in food samples has
been developed. The assay was made possible by using avian antibodies with high-
binding affinity for glyphosate. The matrix effect was found with few food samples.
Microwells immobilised with antigen and blocked could be stored at 48C. These
microwells could be used for routine assay. The assay involved two incubation steps
after which the plate was ready to generate the signal. The assay is very easy to
perform in a microwell plate, the number of wells to be chosen being dependent on
the number of samples. It is not time-consuming and an operator can analyse and
Table 1a. Comparison of immunoassay with HPLC � immunoassay data.
Immunoassay
with food
matrix
(ng/ml)
Concentration of glyphosate (ng/ml)
2 5 10 50 100 250
Turmeric 1.7090.20 4.5090.21 12.3290.02 48.4490.02 82.6090.13 238.3690.15
Chili 1.7691.74 3.8690.16 8.3192.24 46.3291.17 53.4893.24 149.8093.32
Tea (Assam) 1.4992.14 4.4693.20 7.9591.36 43.2190.21 84.3493.14 206.8191.13
Coriander 1.0790.52 4.2290.91 7.9690.94 46.8290.22 80.8293.20 200.3190.92
Coffee 0.1890.22 1.0290.31 1.3291.70 14.6493.20 30.3191.40 140.4093.60
Tea (Ooty) 1.0190.22 3.2891.50 6.3094.10 35.2093.60 70.8091.17 202.4093.95
Rice 1.4393.20 4.3592.10 8.6092.40 44.3190.22 83.4493.24 240.5891.50
Ginger 0.2291.50 0.3690.15 0.7690.22 4.1290.13 6.0291.74 13.8492.90
Notes: Samples were collected as described in experimental sections. Values were a mean of triplicatedeterminations 9 SD. Competitive immunoassays were performed as described in the experimentalsection.
Table 1b. Comparison of immunoassay with HPLC � HPLC data.
Concentration of glyphosate (ng/ml)
HPLC with
food matrix
(ng/ml) 2 5 10 50 100 250
Turmeric 1.8390.16 4.8590.21 9.5490.92 48.1591.36 100.0090.22 241.3891.74
Chili 1.2293.20 3.8290.91 8.3190.15 40.7492.24 75.8391.40 165.3691.50
Tea (Assam) 2.0090.22 4.7190.31 8.6293.20 45.6890.21 99.5891.17 243.7392.60
Coriander 1.8391.50 5.0090.22 9.0891.17 46.6190.22 101.6791.74 244.5190.52
Coffee 2.0992.10 4.8591.74 10.0091.32 43.5293.14 96.6791.91 240.6092.10
Tea (Ooty) 1.9192.20 4.7191.17 9.0892.20 47.5391.13 100.4292.20 244.5191.50
Rice 1.8391.70 4.7191.50 9.2391.80 47.2293.60 95.8392.14 242.9592.14
Ginger 1.0490.31 3.2491.13 6.3190.91 32.7291.40 82.5091.24 165.3691.16
Notes: Samples were collected as described in experimental sections. Values were a mean of triplicatedeterminations 9 SD. Competitive immunoassays were performed as described in the experimentalsection.
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obtain results in less than two hours. It is able to detect glyphosate in food samples at
a concentration as low as 2 ng mL�1. The assay was comparable with HPLC and
future studies will include the exploitation of this approach to other food samples in
the presence of other contaminating pesticides. The stability of IgY being very good
at �208C, single batch preparation of antibodies could be used for a longer time.
Acknowledgements
The authors thank the Director, CFTRI, Mysore, for his encouragement and providingfacilities in the present work. Ms A. Arul Selvi is grateful to the Council of Scientific andIndustrial Research (CSIR) for Senior Research Fellowships. The Government of India isgratefully acknowledged for providing financial support under the Networking project(NWP). The authors are grateful to Dr Muthukumar for his help during immunisation ofpoultry and Mr Akmal Pasha for his help during preparation of conjugates.
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