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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code PS2520

2. Project title

Development of a Portable Biosensor Device for the Determination of Pesticides in Foods

3. Contractororganisation(s)

Central Science LaboratorySand HuttonYorkYO41 1LZ

54. Total Defra project costs £ 29,977

5. Project: start date................ 01 April 2004

end date................. 31 March 2005

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

The main objective of this project was to assess the feasibility of the development of a user-friendly, low cost, portable biosensor device to permit the screening for, or determination of, significant pesticide residues at the point of production, importation, storage or sale of foodstuffs.

Panels of monoclonal antibodies were produced and screened against 25 characterised pesticide (hapten) protein conjugates for the following pesticides; acephate, methamidophos, methomyl and monocrotophos. Antibody panels generally had high affinity towards the carrier proteins, or linkers. Several antibodies appeared to be specific for methamidophos and methomyl, however these could not be inhibited in a competitive ELISA, using varying concentrations of active ingredient. The difficulties encountered probably relate to the very small size and simple structures of the original targets. To date, antibodies suitable for inclusion into a lateral flow devices have not been identified although a small number remain to be characterised.

In order to evaluate the biosensor format within the timescale of the project, alternative organophosphate pesticide reagents, directed against chlorpyrifos, were obtained from HortResearch (The Horticulture and Food Research Institute of New Zealand Ltd). These were successfully validated by ELISA and incorporated into an LFD format. This prototype, coupled with a prototype low cost hand held reader has been successfully evaluated as ‘proof of principle’. Its use as a potential low cost semi-quantitative field device was demonstrated using soft fruit and grape samples received at CSL, collected from retail outlets as part of the PRC monitoring programme.

Chlorpyrifos is a relatively easy compound to analyse by GC-MS or GC-FPD. The reporting limits are usually set by PSD are at 0.02 mg/kg, the MRL’s are relatively high at 0.5, 0.5 and 0.2mg/kg respectively for apples, grapes and strawberries. The instrumental methods generally involve lengthy extraction and clean-up procedures and therefore are more time consuming in comparison with the biosensor approach.

The PRC samples had already been cryogenically milled to produce homogeneous material. The homogeneous material was then sub-sampled and stored at –20°C until analysis. The GC-MS analysis was carried out by the CSL Pesticide Residue Team, and a sub sample sent for LFD analysis. On receipt, a very simple extraction procedure was adopted, consisting of first vortexing the sample with neat methanol at a 1:1w/v ratio. The sample was then centrifuged, the supernatant removed, and diluted in Phosphate Buffered Saline with Tween (PBSt), to a final dilution of 1:8. 75ul of sample was applied to the sample well of the fully assembled device and allowed to run for 5 minutes. The results were visually assessed and subsequently read with the portable LFD reader. The output from the reader provides densities of target (T) and control (C) lines and a reference (R) point, between the lines. The reading takes less than 3 seconds. The results are presented as "R" minus "T" values and when these from all three matrices correlated with the GC-MS determinations an R2 value of 0.9128 was obtained. When individual

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matrices were compared, the correlation was significantly higher, with grapes strawberries and apples giving R2 values of 0.9956, 0.9517, and 0.951 respectively.

The sensitivity of ELISA and LFD was compared using standards of Chlorpyrifos in 10% Methanol. The IC 50 for the ELISA was determined as 0.156 mg/kg and the LFD 0.2-0.23 mg/kg using the same reagents. Further work to independently validate the LFD is imminent.

The potential of LFD as a portable biosensor device for the determination of pesticides in food, has been demonstrated. A significant limiting factor in the development of further assays is the general lack of availability of suitable monoclonal antibodies. There remains a strategic need to investigate the routine production of diagnostic reagents, particularly for very small molecule targets with few or no modifiable groups. The prototype Chlorpyrifos device needs to be evaluated in a field setting by non-specialist end-users, such as PSD sample collection personnel, and feedback obtained on ease of use as well as compiling a validation data set. A simpler and completely portable extraction method also needs to be devised and again feedback obtained from potential end-users.

The authors believe that simple low-cost devices, such as that described here, could play an important role in targeting official surveillance activities as well as providing a tool for the agriculture and food industries. One could imagine "positive release" schemes whereby growers and producers check for significant residues before dispatching consignments of produce to retailers. Simple low cost semi-quantitative devices may also have a role to play in IPM schemes where growers could monitor actual pesticide residues in crops and make more informed decisions about any subsequent applications. Considerable interest in the use of the technology has already been expressed by retailers, grower organisations and agrochemical companies.

Project Report to Defra

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8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Synthesis of pesticides conjugates

It is usually straightforward to develop antibodies to substances that are both antigenic and immunogenic; such as whole cells, viruses, and macromolecules (proteins, complex polysaccharides, lipids, polynucleotides). Compared to this, development of antibodies for small-molecule compounds (termed haptens) needs derivatisation and synthesis of immunogens. This is because haptens of molecular weights lower than 2000 Dalton are generally non-immunogenic, and they need to be chemically linked to a macromolecule carrier (usually a protein) to make immunogens for use in antibody production. Usually haptens have one or more functional groups that can be modified and covalently linked to a carrier macromolecule, usually through a ‘spacer’ moiety. Many pesticides, however, do not have any easily modifiable functional group; and lengthy derivatisations steps are often needed to synthesis immunogens for them.

Acephate(M.Wt. 183.16)

Methamidophos (M.Wt. 141.12)

Monocrotophos (M.Wt. 223.16)

Methomyl(M.Wt. 192.23)

Glyphosate (M.Wt. 169.07)

Figure 1: Chemical structures of the pesticides included in the study,

Five pesticides selected for the study included three OPs (methamidophos, acephate, monocrotophos), one carbamate (methomyl) and a glycine derivative (glyphosate). All of the pesticides are small-molecule non-immunogenic haptens. The first stage of the study, therefore, involved synthesis of chemical conjugates with carrier proteins. A number of methods were used for preparing the conjugates to maximise the chances of making immuogens, and appropriate length ‘spacer’ moieties were provided between the carrier-hapten molecules where possible, to enhance the exposure of the latter to the immune system. In total, 25 conjugates were prepared for use in immunisations and/or assay development. Conjugation methods chosen were those that did not involve lengthy chemical modifications of the haptens and those which have been previously successfully used at CSL for other small molecule haptens. The conjugation methods chosen were those that involved the use of readily available reagents and bifunctional linkers.

1. The primary amine (-NH2) functional group in methamidophos and the carboxyl (–COOH) functional group in glyphosate were modified and linked with carrier proteins using methods described in detail by Chaudhry (2000). In brief, the methods used to activate primary amine included gluteraldehyde, and a commercially available bifunctional linker DSP (Lomant’s reagent, Pierce Perbio) that also provides a 12Å spacer moiety. The activation of the carboxyl group of glyphosate was carried out by EDC/NHS and linking to proteins, or adding an amine group by reacting with ethylenediamine and then linking to proteins via DSP or gluteraldehyde. Using these linking strategies, a number of antibodies have successfully been raised at CSL to small molecule haptens such as antibiotics and the mycotoxin ochratoxin-A.

2. The remaining three pesticides (acephate, monocrotophos, methomyl) were more difficult to conjugate as they contained a secondary amine (-NH-) group, which is not very reactive. Apart from lengthy derivatisation procedures, only the linkers that contain aldehyde or ketone moieties are known to react to some extent with

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secondary amines. The conjugates of these pesticides with carrier proteins were, therefore, prepared using gluteraldehyde, as no other suitable aldehyde or ketone linker is commercially available. Attempts were also made to synthesise conjugates of these three pesticides using succinic anhydride as a linker.

3. Conjugates of all five pesticides were also prepared using a commercially available bifunctional photoactivatable linker, sulfo-HSAB (Pierce Perbio). This class of linkers does not cause any substantial alterations in the chemical structure of haptens, as one end of the linker is reacted with a protein (under dark-room conditions), and the other end is then reacted non-specifically to haptens upon activation by UV light. The use of this method for linking haptens has enabled us to successfully produce antibodies to a fungicide, tebuconazole (Danks et al., 2001).

4. The pesticide-protein conjugates were characterised by determining protein concentration using the Coommassie blue method as described by Bradford (1976).

5. The ratios of conjugation (molar ratio between hapten:protein) was determined by a more recent method based on MALDI-TOF analysis. Using this method, the ratio of conjugation was calculated as the difference in molecular weights between a hapten-protein conjugate and the same protein in unconjugated form (see Fig. 2 for examples). In our experience, this method gives a more precise measurement of the conjugation ratio than the method that has been conventionally used and that is based on determining free amine groups in a carrier protein before and after conjugation (Habeeb, 1966). The latter method is prone to artefacts, as it is likely to give a false indication of higher conjugation ratios that may in fact be due to blockage of amine groups due to inter- and intra-protein cross linking that may happen during a conjugation reaction. It was, however, only possible to determine pesticide conjugation ratios with Bovine Serum Albumin (BSA), as other proteins were either a mixture of different molecular weight fragments (chicken-egg albumin) or too large (keyhole limpet hemocyanin) to determine molecular weight by MALDI-TOF.

Figure 2: MALDI TOF Spectra of Bovine Serum Albumin (BSA) and conjugates with methamidophos.

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Table 1: List of pesticide-protein conjugates synthesised.

Conjugate # Pesticide Carrier protein Linking method Protein

ConcentrationConjugation ratio

93

Glyphosate

BSA Sulfo-HSAB 57 mg/ml 1.9

94 BSA Glut 46 mg/ml 6.8

98 BSA DSP 53.5 mg/ml 1.1

95 Cationised-BSA EDC 110 mg/ml 2.4

97 KLH Glut 4 mg/ml ---

99 OVA DSP 40 mg/ml ---

100 OVA Glut 35 mg/ml ---

106

Methamidophos


102 BSA DSP 50 mg/ml 2.9


92 Cationised-BSA Glut insoluble ---

103 OVA DSP 20 mg/ml ---

104 KLH DSP 2.7 mg/ml ---

107

Methomyl



120 BSA Succinic anhydride 15.8 mg/ml 0.9

121 OVA Succinic anhydride 18.6 mg/ml ---

109

Acephate





111

Monocrotophos





Key: BSA = bovine serum albuminOVA = chicken-egg albumin (ovalbumin)KLH = keyhole limpet hemocyanin

Monoclonal antibody production

Panels of monoclonal antibodies were produced using standard protocols (Galfre and Millstein 1981) and cell-lines screened as described below.

Plate trapped antigen format ELISA

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96 well plates were coated with 100μl pesticide conjugate (in 15mM Na2CO3, 35mM NaHCO3, pH 9.6), and incubated at 33oC for 2 hours. Plates were washed 3 times with PBST (20mM sodium phosphate, 150mM sodium chloride pH 7.4 containing 0.05% Tween 20) and blocked with 250μl 5% milk powder in PBST for 1 hour at 33oC. After washing 3 times with PBST, 100μl antibody (tissue culture supernatant diluted 1:1 with PBST/0.2%BSA or polyclonal test sera at various dilutions) was added to each well, for 1 hour at 33 oC. After washing plates 3 times with PBST, 100μl goat anti-mouse IgG alkaline phosphatase (Sigma, A-4312) was added at 1:4000 in PBST/0.2%BSA for 1 hour at 33oC. After washing plates 3 times with PBST, 100μl of pNPP solution (1mg/ml in 10% diethanolamine, 0.02% MgCl2, pH 9.8) was added to each well. Plates were read after 1 hour at room temperature, at 405nm.

Inhibition ELISA96 well plates (Nunc, maxisorp, 442404) were coated with 100μl pesticide conjugate as described in the PTA methods. Plates were washed 3 times with PBST and blocked with 250μl 5% milk powder in PBST for 1 hour at 33oC. Plates were washed 3 times with PBST. In a separate plate, 100ul of pesticide at a range of dilutions, with 100μl of purified monoclonal antibody (1:1000 in PBST/0.2% BSA) for 1 hour at 33oC was pre-incubated. 100μl of the pre-incubated pesticide/antibody mix was added to the coated plate and incubated at 33 oC for 1 hour. Plates were washed 3 times with PBST and 100ul goat anti-mouse IgG alkaline phosphatase (Sigma A-4312) at 1:4000 in PBST/0.2%BSA added and incubated for 1 hour at 33oC. After washing plates 3 times with PBST, 100μl of pNPP solution was added to each well. Plates are read after 1 hour at room temperature, at 405nm.

MethamidophosPanels of monoclonal antibodies were raised against a mixture of conjugates 102, 105 and 106, all methamidophos: BSA conjugates, but each using different conjugation chemistry.

After extensive screening with all available conjugates all antibodies raised from fusion 288 were positive for the BSA conjugates of the methamidophos pesticides, but also to BSA.

Figure 3: Screening data from fusion 288.

All 6 hybridomas were cloned and evaluated further, using various blocking techniques to reduce the cross reactivity with BSA, followed by inhibition ELISAs, but no inhibition was observed using the pesticide at a concentration of 1000ppm. The evaluation was therefore stopped, as none of the antibodies produced were specific for methamidophos.

From fusion 333, 12 monoclonal antibodies were selected and evaluated, 2 of which appeared promising and were evaluated fully. These antibodies do not detect any BSA conjugates of methamidophos (the carrier molecule used in the immunogen) but do detect a KLH conjugate which has methamidophos conjugated using the same linker, as that used with the BSA carrier for immunisation and screening. These antibodies are still being evaluated, however to date no free inhibition using methamidophos has been demonstrated. Confirmatory tests to determine whether they are detecting the pesticide or the linker molecule will be done, as the same chemistry was used in the preparation of conjugates 102 and 104. The data is shown below in Figure 4.

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Figure 4: Screening data from fusion 333.

Fusion 337 produced 7 monoclonal antibodies, but all antibodies cross reacted with BSA, the carrier molecule.

In total, 7 monoclonal antibodies were produced from fusion 344, none of which cross-reacted with BSA. Unfortunately, further evaluation resulted in the antibodies being classified as specific for the linker molecule (sulpho-HSAB). Competition ELISAs did not result in inhibition of the antibodies using free methamidophos at 1000ppm (1mg/ml). However, the antibodies all gave good positive ELISA results when screened against conjugates 106 and 109, a methamidophos-BSA (sulpho-HSAB link) and acephate-BSA (sulpho-HSAB link), but not with other conjugates of methamidophos using different linker chemistries. These antibodies were further screened against other unrelated compounds using the same linkage chemistry. All antibodies showed the same pattern, however only 3 are shown below. These antibodies were therefore unsuitable for use in this project.

Figure 5: Screening data from fusion 344, all antibodies specific for sulpho-HSAB.

Acephate

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Panels of monoclonal antibodies were raised against a mixture of conjugates 109, 110 and 125, and polyclonal anti-sera was screened against available conjugates.

The screening of the hybridomas from fusion 307 showed all monoclonal antibodies produced (22 in total) cross react with BSA. Optimisation of the assay by pre-absorbing the monoclonal antibodies with BSA did not result in a reduction of cross reactivity, and as a result no pesticide specific antibodies were produced, as shown below in Figure 6.

Figure 6: Screening data of antibodies produced in fusion 307.

Once again, after extensive screening, all antibodies produced from fusions 317 and 325 (37 in total) cross reacted with BSA. In general the hybridomas produced from fusions 317 and 325 were of unusually poor quality.

Monocrotophos

Panels of monoclonal antibodies were raised against a mixture of conjugates 111, 112 and 121 (all monocrotophos: BSA conjugates), and both the polyclonal sera and all antibodies were screened with all available conjugates.

Figure 7: Polyclonal sera response data with monocrotophos conjugates.

54 hybridomas producing positive antibodies were selected from the 3 fusions, however all monoclonal antibodies produced cross reacted with BSA, the carrier molecule. As a result no antibodies specific for monocrotophos were produced.

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Methomyl

Panels of monoclonal antibodies were raised against a mixture of conjugates 107, 108 and 120. Test sera and all antibodies produced were screened with all available conjugates. Three further panels of antibodies remain to be screened, work which will commence April 2005.

Figure 8: Polyclonal response for methomyl conjugates.

The polyclonal response for test 263.3 showed early promise, with a higher specificity for the conjugates compared to BSA. However, this fusion produced only 21% hybrids (an unusually low number) of a poor quality. Unfortunately, no specific monoclonal antibodies were generated. 4 hybridoma cell lines are still being evaluated, where specific antibodies are in the early stages of development. However, these hybrids are unstable and proving to be extremely problematic.

Figure 9: Screening data of 4 hybridomas from fusion 336 which are still being evaluated.

To date, 5 fusions have been done and in total, 105 hybridoma cell lines were selected for further analysis, but all other antibodies cross-reacted with BSA.

Evaluation of 4 anti-methomyl hybridomas, 1 anti-methamidophos and one anti-acephate monoclonal hybridoma is on-going.

An exhaustive search for a commercial source of polyclonal or monoclonal antibodies for the target pesticides was carried out. A number of commercial and academic sources for the required antibodies were approached and 2 academic groups agreed to supply antibodies for evaluation. However, to date no commercial source for

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antibodies to any of the pesticides could be identified, indicating just how challenging it is to make antibodies to extremely small pesticides.

Evaluation of anti-acephate polyclonal antibodies from Bruce Hammock et al

We received an acephate: BSA conjugate and anti-acephate rabbit polyclonal antibodies from Bruce Hammocks group (Lee et al, 2003). The antibody was evaluated against the supplied conjugate and the acephate conjugates produced at CSL. The published data uses the antibody in an ELISA using an anti-rabbit horseradish peroxidase secondary antibody. Using this system, absorbencies observed were barely above background levels. It was decided not to pursue development using this antibody as the published data did not lead us to believe the antibody would be specific enough for use in a lateral flow device.

Evaluation of antibodies from HortResearch in New Zealand

We received 5 monoclonal anti-OP pesticide antibodies from HortResreach in New Zealand, along with matching conjugates. These antibodies were raised to:

Chlorpyrifos (MW 350Da) Diazinon (MW 304Da) Parathion (MW 291Da) Azinphos (MW 317 Da) Generic OP antibody

All antibodies were evaluated against all the conjugates received, using a chequerboard ELISA format, to determine both the optimum coating concentration and antibody concentration. After optimisation, the antibodies were all subsequently evaluated in a series of competitive ELISAs to determine if inhibition of antibody could be achieved using both the supplied conjugate and free pesticide, plus the concentrations of each required achieving 50% inhibition of the antibody. This data in shown in Figure 10 and table 4 below:

Figure 10: Inhibition of the anti-parathion, anti-chlorpyrifos, anti-diazinon and anti-azinphos antibodies with the corresponding conjugates.

50% inhibition using the conjugates was observed at the following concentrations:

Table 4: IC50 values of antibodies inhibited by the supplied conjugates.

Antibody used Inhibiting conjugate IC50Anti-azinphos Azinphos 3 ppbAnti-diazinon Diazinon 14 ppbAnti-parathion Parathion 39 ppbAnti- Chlorpyrifos Chlorpyrifos 156 ppbAnti-generic OP Chlorpyrifos 3 ppbAnti-generic OP Generic OP conjugate 10 ppb

Pesticide standards were sourced and made up at various dilutions in 10% methanol. Competitive ELISAs were carried out on all of the antibodies to determine the concentration of free pesticide required for 50% inhibition levels. Inhibition of the generic antibody was also tried using methamidophos standards, and methamidophos,

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acephate, monocrotophos, methomyl and Glyphosate conjugates. No inhibition was observed with any of these up to1000 ppm.

Figure 11: Inhibition of the Chlorpyrifos antibody with Chlorpyrifos pesticide.

Lateral Flow Device Developments

Initial LFD development work centered on a ‘generic’ "OP" polyclonal antibody already available at CSL, that reacts with a number of organophosphate (OP) pesticides in ELISA. The aim was to test the suitability of this preparation for incorporation into a prototype LFD-biosensor.

An overview of the development of ‘generic OP’ antibody based LFD is described in the schematic diagram below, Figure 12.

(i) Generic-OP hapten conjugate with BSA was sprayed onto membrane to form parallel test and control lines of anti-rabbit antibody.

(ii) Coloured latex beads were coated with polyclonal antisera (R288) previously raised to a generic OP hapten-BSA conjugate.

Figure 12: Diagrammatic representation of a generic OP Lateral Flow Device.

The biosensor works by applying a test liquid sample, which moves into the device where it mixes with the antibody-latex complex and flows across the membrane. If an OP compound was present, binding would occur with the antibody bound to the coloured latex. This complex would then flow across the membrane and bind with an anti-species antibody forming a visible blue control line. In this scenario, the antibody-latex complex would not bind with the OP conjugate present at the test line, as the specific anti-OP sites on the latex would be saturated with OP from the test sample.

If a sample did not contain any OP compound, it would flow across the membrane and allow binding with the OP-BSA conjugate test line, as well as with the anti-species control line, forming two visible blue lines.

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Thus appearance of both control and test lines would indicate that the test has been performed successfully. The appearance of a test line would only indicate a negative sample (i.e. analyte of interest was not present), whereas the absence of a test line would indicate a positive sample.

Initial investigations into the development of an OP LFD were carried out by spraying two generic-OP conjugates (with BSA and OVA) on to a membrane at 1, 0.1 and 0.01 mg/ml concentrations. The polyclonal antiserum (R288) was coated to coloured latex beads at 2, 10 and 40 mg/ml. Wet assay testing allows for preliminary evaluations of the spraying and coating procedures. Testing revealed that only control line development was achieved. No test line on either of the conjugates sprayed membranes was formed at even very high latex coating concentrations.

Polyclonal antisera are composed of a mixture of antibodies, including those that are reactive to carrier proteins. A strong reaction to carrier protein, nevertheless, causes major problems to the sensitivity of a biosensor, as it masks the reaction of analyte-specific (anti-OP in this case) antibodies. Furthermore, the latex coating procedure uses BSA in a blocking step, and thus the presence of anti-BSA antibodies in the tested antiserum was giving a very high background that was also masking the reaction of OP-specific antibodies.

To overcome these difficulties, and produce a ‘proof of principle’ device we sourced a range of monoclonal antibodies from HortResearch, New Zealand, and amongst these was a suitable monoclonal antibody that specifically detects the OP pesticide Chlorpyrifos.

Development and validation of the Chlorpyrifos LFD

A Chlorpyrifos specific LFD was developed as previously described for the generic OP LFD, but a specific Chlorpyrifos monoclonal and a Chlorpyrifos-BSA conjugate were substituted. The test effectively runs with the addition of 10-12.5% methanol solutions, in PBSt.

The device is a competitive LFD, and two lines indicate a negative sample, or a sample below the limit of detection. One line (control only) indicates a high positive sample, causing complete ‘wipe-out’ of the target line. In between is a gradient of line densities which are often difficult to assess by eye, but can be accurately determined using a portable densitometry reader, designed specifically for CSL devices. A typical response is shown in the Figure 13 below.

Figure 13: Chlorpyrifos LFD measuring 3 PRC Grape Samples in duplicate.

The sensitivity of the LFD in buffer, was determined. A serial dilution of Chlorpyrifos standards, diluted in 10% methanol and PBSt from a range of 1000 mg/kg to 0.001 mg/kg, was produced and 75ul of standard was added to each device, in duplicate (Set 1 & 2) and the sample was allowed to run for 5 or 10 minutes. The results were visually assessed and subsequently read with the portable LFD reader. The output from the reader provides densities of target (T) and control (C) lines and a reference (R) point, between the lines and takes less than 3 seconds. The results from these reading are shown as in Figures 14 and 15, as absolute reader value (R-T) and as percentage inhibition respectively, using buffer only as a reference.

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Figure 14: LFD reader values comparing duplicate devices at 2 time intervals over a range of standards.

Figure 15: Percentage Inhibition calculation for mean of duplicates at two time points over a range of standards.

From the results it can be seen that there is good correlation between duplicate devices, and that there is no significant difference in IC50 values, in relation to time. The IC50 in solvent was determined as 0.22 mg/kg. This is comparable to the ELISA determination previously reported, at 0.156 mg/kg. It is always advisable to read LFD tests within a set time limit to avoid any background development. The results show that the Chlorpyrifos LFD can be read at any time between 5 and 10 minutes following sample addition, and timing is not critical, a significant advantage when it comes to field applications.

To further validate the Chlorpyrifos LFD, samples were received by the CSL Pesticide Residues Team from the PRC programme. Three samples, a high, low and blank for three different matrices, apple, strawberry and grape, where provide blind for LFD analysis. The PRC samples had already been cryogenically milled to produce homogeneous material. The homogeneous material was then sub-sampled and stored at –20°C until analysis. The GC-MS analysis was carried out by the Pesticide Residue Team, and a sub- sample sent for LFD analysis. On receipt, a very simple extraction procedure was adopted, consisting of first vortexing the sample with neat methanol at a 1:1w/v ratio. The sample was then centrifuged at 12,000 rpm for 1 minute, the supernatant removed, and diluted in Phosphate Buffered Saline with Tween (PBSt), to a final dilution of 1:8. 75ul of sample was applied to the sample well of the fully assembled device and allowed to run for 5 minutes. The results were analysed using the reader and R-T values presented (Figure 16).

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0

50

100

150

200

250

0.0001 0.001 0.01 0.1 1 10 100

Chlorpyrifos Standard (mg/kg)

LFD

Rea

der V

alue

(R

-T)

Set 1 @ 5minsSet 2 @ 5minsSet 1 @ 10minsSet 2 @ 10mins

-20

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Chlorpyriphos Standards (mg/kg)

Perc

enta

ge In

hibi

tion

5 minutes10 minutes

50% Inhibition

Figure 16: Comparison of determinations, in duplicate, for a variety of sample matrices.

The general trend for all samples was as required, low target line densities, due to increased inhibition of the target line for free Chlorpyrifos in the samples. The grape sample with the greatest residue content had been determined by GC-MS as above the MRL (1.1mg/kg), this was clearly seen as an almost complete ‘wipe-out’ of the target line (R-T <19). The additional samples gave an overall good correlation with the GC-MS determination, as shown in Figure 17. However there is clear matrix effect with the apple samples, with a relatively high reader value for the highest sample (0.2mg/kg) compared to the highest strawberry sample (0.12mg/kg). All blank samples were determined correctly. The correlation between LFD and GC-MS was the highest for the grape sample R2= 0.9956. Further work is required to optimise the method for alternative matrices.

It is proposed that a further blind validation should be performed at another recognised PRC analytical laboratory, using the same samples used in this study.

Figure 17: Correlation between GC-MS and LFD, with all 9 samples, 3 matrices.

Reproducibility (batch to batch and test to test), stability and defined limits of detection, in different matrices, should be determined, once additional reagents are received from HortResearch, (currently under negotiation). Depending on the cost of any necessary reagents, it is envisaged that devices could be manufactured (by CSL) at a cost of about £3 per test. The associated portable reader is likely to cost around £300. Data from this project may be used to support two CSL Defra patent applications relating to simple pesticide residue determination using LFDs.

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y = -0.5396Ln(x) + 2.4542R2 = 0.9128

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120

LFD Reader Value (R-T)

GC

-MS

(mg/

kg)

0

20

40

60

80

100

120

53373a 53373b 51562a 51562b 48029a 48029b 47411a 47411b 50007a 50007b 50369a 50369b 50272a 50272b 54071a 54071b 53372a 53372b

Sample Number, (replicate a or b)

LFD

Rea

der V

alue

(R-T

)

Apple matrix Strawberry matrix Grape matrix

GC-M

S Result - Blank

GC-M

S Result - Blank

GC-M

S Result - Blank

GC-M

S Result - Blank

GC-M

S Result - Blank

GC-M

S Result - Blank

GC-M

S Result - 0.02 mg/kg

GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


GC-M


The potential of LFD as a portable biosensor device for the determination of pesticides in food, has been demonstrated. A significant limiting factor in the development of further assays is the general lack of availability of suitable monoclonal antibodies. There remains a strategic need to investigate the routine production of diagnostic reagents, particularly for very small molecule targets with few or no modifiable groups. The prototype Chlorpyrifos device should be evaluated in a field setting by non-specialist end-users, such as PSD sample collection personnel, and feedback obtained on ease of use as well as to compile a validation data set. A simpler and completely portable extraction method also needs to be devised and again feedback obtained from potential end-users.

The authors believe that simple low-cost devices, such as that described here, could play an important role in targeting official surveillance activities as well as providing a tool for the agriculture and food industries. One could imagine "positive release" schemes whereby growers and producers check for significant residues before dispatching consignments of produce to retailers. Simple low cost semi-quantitative devices may also have a role to play in IPM schemes where growers could monitor actual pesticide residues in crops and make more informed decisions about any subsequent applications. Considerable interest in the use of the technology has already been expressed by retailers, grower organisations and agrochemical companies.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Lee, J.K., Ahn, K.C., Stoutamire, D.W., Gee, S.J., Hammock, B.D. (2003) Development of an Enzyme linked Immunosorbent Assay for the Detection of the Organophosphorous Insecticide Acephate”, J. Agric. Food Chem. 2003, 51, 3695-3703.

Danks, C., Chaudhry, M.Q., Parker, L., Barker I. and Banks J.N. (2001) Development and validation of an immunoassay for the determination of tebuconazole residues in cereal crops, Food and Agricultural Immunology 13: 151-159.

Chaudhry, M.Q. (2000) Chapter 6: Standards and Immunogens, in Immunoassays: A Practical Approach, pp 165-185, Gosling, J.P. (Ed.), Oxford University Press, ISBN 0-19-963710-5.

Kane, M. and Banks, J. N. (2000) Chapter 3: Raising monoclonal antibodies, pp 19-58 in Immunoassay: A Practical Approach, Gosling, J.P. (Ed.), Oxford University Press, ISBN 0-19-963710-5.

Banks, J.N., Chaudhry, M.Q., Matthews, W.A., Haverly, M., Watkins, T. and Northway, B.J. (1998) Production and characterisation of polyclonal antibodies to the common moiety of some organophosphorus pesticides and the development of a generic type ELISA, Food and Agricultural Immunology 10(4): 349-361.

Matthews, W.A., Banks, J.N. and Chaudhry, M.Q. (1994) Methods, Agents and Kits for detection of organophosphorus compounds, World Patent Application WO 94/27149.

Danks, C., Ostoja-Starzeweska, S. and Banks, J. (2003) The development of a lateral flow device for the discrimination of OTA producing and non-producing fungi. Aspects of Applied Biology 68, 21-28.

Itak, J.A., Selisker, M.Y., Root, C.D., Herzog, D.P., (1996) Validation of an immunoassay for methomyl in water and dislodged residues on grape leaves. Bull. Environ. Contam. Toxic 57, 270-277.

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