Patent ID: 12188933

EXAMPLES

Work conducted at The University of the West of England (UWE), Bristol established a strategy to develop a rapid hand held in field biosensor to detect Cocoa Swollen Shoot Virus (CSSV) infection ofTheobroma cocoa.

Evaluation of Florescent Reporter

Quantum dots (QDs) were evaluated as a fluorescent reporter and were shown to fluoresce at specific wavelengths dependant on the size of the quantum dots (FIG.2). Pilot work showed the proof of concept to use streptavidin tagged quantum dots binding biotin conjugated antibody captured on a rapid flow through paper based cassette (FIG.3). Other membrane types evaluated include PVDF, Polycarbonate and mixed Cellulose Ester (Data not shown).

Three ESELog ESML10-MB-3018 confocal fluorescence detectors were commissioned and produced with two excitation wavelengths:

E1—365 nm, E2—660 nm and two emission filters D1—625 nm, D2—720 nm.

On evaluation it was found that the positioning of the quantum dots on the membrane gave maximum readings when positioned off centre (seeFIGS.4Aand B).

Sequence Analysis of Published CSSV Sequences to Establish the ORF3 Region of CSSV Associated with the Viral Coat Protein.

Antibodies raised to virus purified from plant leaves have shown high background values in immunoassays. A strategy was developed to determine the CSSV coat protein sequence by alignment with other similar viral sequences and to produce a recombinant CSSV coat protein antigen (CSSV-CP-01). This would then be used to generate recombinant binding reagents e.g. Aptamers, Monoclonal Antibodies or Affimers, by phage display; this would therefore limit background interference as no plant material was used in the selection process. Research was performed into the costs, requirements and risk of the selection process, which led to a final choice of generation of recombinant Monoclonal Antibodies by phage display.

Tables 1 & 2 show reagents and equipment produced.

TABLE 1Reagents produced.Reagents producedDetailsRecombinant CSSV coat protein antigen/Based on x5 publishedCSSV-CP-01. Two batches weresequencessynthesised, purified and used in theexperiments shown CSSV-CP-01a andCSSV-CP-01bRecombinant CSSV coat protein antigen/Based on sequences fromCSSV-CP-02.Muller et al., 2018 andChingandu et al., 2017Recombinant monoclonal antibodiesProduced by phage displayPolyclonal antibody produced in rabbitsInoculum CSSV-CP-01a

TABLE 2Equipment producedEquipment producedDetailsAdditional informationESELog fluorimetersX3 producedNarrow beamRange of emissionsdetected3D printed holder for14 mm and 10Blocks natural light fromfluorimeter and cassettemm basescontributing to readings
Optimisation of Extraction of Virus from Plant Material.

A supply of CSSV infected and non-infectedTheobroma cocoaplants was established from Reading university and were housed in the Envirotron at UWE. A range of buffers and methods of extraction were evaluated using qPCR to determine success.

Method of Extraction from Leaves

The leaves were harvested from CSSV infected and uninfected cocoa plants and roughly chopped with scissors into 0.5 cm pieces. 60 mg leaf tissue was weighed and placed in a 7 ml bijoux or Eppendorf tube containing 10×4 mm ball bearings and 2 ml 0.1M Phosphate buffer, pH 7.2. The vessel was shaken for approximately 2 minutes. The solution was passed through a sieve layer with a minimum pore size 0.1 um and collected. The solid dry plant tissue retained by the sieve layer was discarded and ball bearings recovered and washed. The plant sample was then added to the biosensor.

A table showing the effect of different amounts of leaf tissue, size of ball bearings and volume of buffer is shown inFIG.5.

Method of TaqMan qPCR

Leaf tissue was extracted in on the day of collection and DNA purified immediately from a 20 μl sample using Qiagen Plant Dneasy mini kit. The kit was used following the manufactures instructions but omitted the RNAse treatment step and eluted sample twice in the same 100 μl buffer at the end to enrich. Samples were stored at −20° C. until use.

TaqMan qPCR was then performed on the DNA samples using a CSSV primer/probe and a plant genomic probe and the Sensifast No RoxMaster Mix (Bioline Cat. BIO-98005). All samples were run in triplicate.

The CSSV and plant genomic probe/primer mix comprised of:

2.5 μl (100 μM) probe10 μl (100 μM) forward primer10 μl (100 μM) forward primer77.5 μl elution buffer (Qiagen)CSSV ORF3 primers and probeF-74:{SEQ ID NO: 32)5′-CTGAAGCGAGTAGGCAACAA-3′R-151:(SEQ ID NO: 33)5′-CAGTCCAAGGGATGGACTCT-3′P-129:(SEQ ID NO: 34)5′-TCCATCAGGTTGCCATGGCA-3′ (5′Fam-3′Tamra)Primers & probe for nuclear marker in flankingregion of single copyT.cacaomicrosatellitemarker mTcCIR25F-mTcCIR25:(SEQ ID NO: 35)5′-CAGATAAGGAAAGGTGGAGTTTGG-3′R-mTcCIR25:(SEQ ID NO: 36)5′-CAAGAATGTCTCCTACATTCACTACG-3′P-mTcCIR25:(SEQ ID NO: 37)5′-TTCCCGTAAGCTTCGTCCCAGATGC-3′ (5′Fam-3′Tamra).

Each PCR reaction comprised of: 0.8 μl probe/primer mix10 μl Mastermix (Bioline)4.2 μl nuclease free water5 μl DNA

The reactions were run on a Rotor Gene Q instrument. Hold 95° 5′, Cycle: 95° C. 10 s (acquiring to cycling A), 60° C. 45 s (acquiring to cycling B). Cycle is repeated 60 times.

The number of CSSV copies were determined in each sample by comparison to a synthetic CSSV references DNA oligomer of know copy number and the CSSV copy number per cell was estimated from the ratio of CSSV copy to plant cell copy number.

Assay Development

Evaluation of the Interaction of Recombinant Antigen CSSV-CP-01 and Recombinant Antibodies.

Testing ofTheobroma cacoaPlant Samples/Extracts by Competitive ELISA

Materials:

Coating buffer: 0.1M sodium carbonate buffer pH 8.6Sodium carbonate MW 106, so 1.06 g/100 mis dH2OSodium hydrogen carbonate MW 84.01, so 0.86 g/100 mis dH2O, Mix to pH 8.6Blocking buffer PBS, 1% Bovine serum albumin (Sigma A7030, #SLBL4277V)Wash buffer (PBS, 0.05% tween 20)Antibody dilution buffer (PBS, 0.05% tween 20, 0.1% BSA)Tetra methylbenzidine Liquid Substrate (Thermo)Microlon high binding plate (Greiner 655061)CSSVP-CP-01 recombinant fusion protein—Coating concentration 1 to 10 μg/ml
Recombinant ReagentsGoat anti Human IgG F(ab′)2:HRP (Bio-Rad STAR126P)AbD31998.1 anti-CSSV antibody (BioRad) final concentration 50 ng/ml
Plant Samples:Leaves were harvested fromTheobroma cacaoplants in the museum collection at CRIG, Ghana and roughly chopped with scissors into 0.5 cm pieces. 60 mg Leaf tissue was weighed and placed in a 7 ml bijoux or Eppendorf tube containing 10×4 mm ball bearings and 2 mls of 0.1M Phosphate buffer B pH7.2 was added and the vessel shaken for approximately 5 minutes.
Procedure:1. Allow all reagents to reach room temperature before use. Mix all liquids gently prior to use.2. Prepare CSSVP-CP-01 Antigen, diluted to 1-10 μg/ml in coating buffer.3. Take immunoassay plate. Add 50 μl of prepared solution to wells. Cover plate and Incubate at 4° C. for 18 hours in a humid box.4. Decant samples and discard. Wash wells 3 times in Wash buffer using a squirt bottle. Tap plate gently on tissue following every wash to remove all liquid.5. Fill the wells with 200 μl Blocking buffer. Incubate at room temperature for 2 hours.6. Prepare recombinant monoclonal antibody at 200 ng/ml in antibody dilution buffer ie. at 2× final concentration.7. Prepare competing moieties at 2× final concentration in antibody dilution buffer:i. Plant sample at 1/5ii. Plant sample at 1/50iii. Plant sample at 1/500Recombinant antigen at 1.25 and 0 μg/ml.Equal volumes of antibody and competing recombinant antigen, plant extract or buffer were mixed in an Eppendorf tube and incubated for 30 mins at room temperature.8. Decant blocking buffer and discard. Wash wells 4 times in Wash buffer. Add 50 μl of primary antibody/competitor to appropriate well. Cover plate and incubate at room temperature for 60 minutes.9. Wash wells 5 times in Wash buffer.10. Add 50 μl of secondary antibody Goat anti Human IgG F(ab′)2:HRP at 1/2500 in antibody dilution buffer to each well. Cover plate and incubate at room temperature for 60 minutes.11. Wash wells 5 times in Wash buffer.12. Add 100 μl 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate to the wells. Incubate for 10 minutes at room temperature in the dark.13. Add 100 μl 1M Sulphuric Acid14. Read absorbance of wells at 450 nm in a microtitre plate reader
Evaluation of Candidate Recombinant Monoclonal Antibodies

Fifteen candidate monoclonal antibodies were supplied and evaluated for their ability to bind to recombinant antigen by ELISA (seeFIG.7A). A competitive ELISA was then performed with CSSV positive and negative leaf samples (seeFIG.7B). Candidate antibodies were selected for biotinylation based on results with leaf extracts. Antibodies that bound to CSSV-CP-01 antigen that were competed by CSSV positive leaf extracts, but not by CSSV negative leaf extracts were identified. Recombinant antibodies were selected for biotinylation and used in the Biosensor development including AbD31024.2, AbD31988.1, AbD31999.1 and AbD31998.1.

The results of replicate ELISA experiments for the selected antibodies, across a range of plant extract dilutions are shown in Tables 3 andFIGS.8and9. A strong reproducible dose response was shown by all antibodies. A summary of the selectivity and specificity of each candidate recombinant monoclonal antibody is shown in Table 4.

TABLE 3Competitive ELISA results of qPCR confirmed CSSV infected plant samplesand binding of recombinant antigen and recombinant monoclonal antibodies.Percentage Inhibition %CompetitorNo PrimaryrAntibodyAbD31024.2AbD31025.2AbD31988.1AbD31994.1AbD31999.1controlRec. Antigen24−4.538.926.311.72.9Pos extract5.3−12.415.95.040.81.81/1000Pos extract16.3−12.446.95.069.112.31/100Pos extract70.1−19.885.219.578.517.81/10Neg extract−3.8−32.4−1.60.99−2.714.91/1000neg extract−8.2−12.1−1.75.0−17.515.91/100Neg extract−29−42.1−4214.6−63.920.31/10

TABLE 4Summary of the selectivity and specificity of recombinantmonoclonal antibodies binding to CSSV and plant tissue.CompetitionCompetitionInteractionby CSSVby Non-WithInfectedInfectedAssayAntibodyCSSV-CP-01plant tissue*plant tissue*CandidateYYNYAbD31025.2YNNNAbD31986.1YNNNYYNYAbD31989.1YYNYAbD31990.1YNNNAbD31991.1YNNNAbD31992.1YNNNAbD31993.1YNNNAbD31994.1YYYNAbD31995.1YYYNAbD31996.1YYNYAbD31997.1YYNYYYNYYYNY*Above background.Antibodies highlighted in italics are assay candidates. Antibodies in bold were selected for progression, the amino acid and DNA sequences of antibodies AbD31998.1, AbD31999.1 and AbD31988.1 are shown SEQ ID NOs: 5-31.
Development of Competitive Flow Through Assay—CSSV Biosensor.

The first available biotinylated candidate antibody AbD31024.3 was used in the development of the biosensor in terms of materials, timing, buffers, QDs and detection with the ESElog fluorimeter.

Testing of Plant Samples/Extracts Using the Biosensor

Materials

MDI membrane 0.8μ CLW-040-SH34 (DL191/3 #LD191917G)MDI Absorbent pads AP080 of 24.5 mm×36 mmTesco disposable nappy—absorbent layerCSSVP-CP-01 recombinant fusion proteinAbD31998.5 biotinylated 0.65 mg/mlQdot® 605 nm streptavidin conjugate (life technologies cat. Q10101MP lot #1826419)Phosphate buffered saline pH 7.3 (PBS)Non-fat dried milk (Marvel)
Method for Biosensor Competition Assay1. The membrane was placed onto a foil (non-porous) solid support which enabled antibody to be dried to the membrane. The membrane was cut to fit into a cassette and was marked with a pencil so that placement of the dots could be navigated in to the read area. CSSV antigen was diluted in PBS to 100 μg/ml. 10 μls was spotted onto the paper cast Nitrocellulose Membrane of pore size <1 μm and left to air dry at room temperature for 15 mins.2. The membranes were then blocked in 5% skimmed milk in PBS for 2 hours at room temperature and washed 3 times in PBS. Membranes were stored at 4° C. for 24 hours.3. Each membrane was assembled onto a stack of 2 MDI Absorbent pads AP080, and a single layer of absorbent material cut to fill the entire area of the cassette.

Biotinylated recombinant monoclonal antibody was diluted to 8 μg/ml in PBS (2× final concentration).4. Competing test samples were diluted in PBS to 2× final concentration. Plant samples were therefore diluted 1/5, 1/50, 1/500 and 1 μg recombinant antigen was diluted in 100 μl PBS. Sufficient quantities were prepared such that all tests were repeated in triplicate. 100 μl competitor or control PBS was mixed with 100 μl of recombinant monoclonal antibody and left at room temp for 30 mins to pre-incubate prior to assay. All combinations were prepared in triplicate.5. 200 μl of antibody/competitor was dripped slowly onto the centre of the spot and allowed to flow through.6. qDots 605 streptavidin conjugate were diluted to 10 nM in PBS and 10 μl was applied and allowed to flow through. This was followed by 2×PBS washes of 500 μl.7. The fluorescent signal of bound Qdots were measured at an excitation wavelength of 365 nm and emission of 625 nm using the Fluorescence Detector (E1D1) at a distance of 1.4 cm.

Detection of binding of monoclonal antibodies to antigen CSSV-CP-01 using biotinylated antibody AbD31024.3 is shown inFIGS.10and11.

Evaluation of the other candidate recombinant monoclonal antibodies showed AbD31988.5 to give the highest signal and clearest dose response binding to CSSV-CP-01 as shown inFIG.12. The reduced binding of antibody in the presence of free competing recombinant antigen CSSV-CP-01 is shown in Table 5 andFIGS.13and14.FIG.16also shows competition by CSSV positive plant extract.

TABLE 5CSSV Biosensor results showing competition by two concentrationsof competing recombinant antigen CSSV-CP-01 (n = 3)ConcentrationRepRepRepof CSSV-CP1.v1123MeanSDSECV500 ng rec. Ag.1575152013481481118688.0100 ng rec. Ag.187422141859198220011610No competition250019542500231831518214

A compilation of three separate Biosensor experiments is shown in Tables 6-8. The results shown are the competition observed by recombinant antigen in three separate experiments using two different batches of CSSV-CP-01a and CSSV-CP-01b.

TABLE 6CSSV Biosensor results showing competition by recombinantantigen in three separate experiments using two differentbatches of CSSV-CP-01a, b (n = 3 in each experiment)Fluorescent signalSample123MeanSDSECV500 ng rec. Ag.1570136015921507128428.4CSSV-CP-01bNo competition151620052378196643214421.9Background100811031134108165216

TABLE 7UWE, CSSV-CP-01a, CSSV positive plantextract n = 3, QDots - 705 nmFluorescent signalSampleRep 1Rep 2Rep 3MeanSDSECVPositive extract33729833032120126.51/20500 ng rec. Ag.3643533603595.53.21.5CSSV-CP-01aNo competitor44148242644928.916.86.4

TABLE 8Competitive Flow through assay to demonstrate competitionby CSSV-CP-01a antigen n = 3, QDots - 605 nmConcentrationRepRepRepof CSSV-CP1.v1123MeanSDSECV1000 ng rec. Ag.211521751573195433119116.9500 ng rec. Ag.1575152013481481118687.9100 ng rec. Ag.187422141859198220011610.1No competition250019542500231831518213.5
Report on the Evaluation of Reagents and Immunoassays to Detect Cocoa Swollen Shoot Virus (CSSV) at the Cocoa Research Centre of Ghana (CRIG).

Day 1. Familiarisation with laboratories, unpack reagents and materials, stage 1 of ELISA.

Day 2. Collection of leaf samples 1-15 from museum collection at CRIG includes three strains of CSSV, New Juaben, Kpeve and Nsaba. All samples collected were photographed (not shown).

Uninfected plants samples A-D, were collected from a different part of the CRIG compound.

An accurate amount of each sample was weighed and the leaf tissue extracted. Plant extracts were stored at 4° C.

The ELISA assay was completed on 15 plant extracts, 1-15 and 4 CSSV negative plant extracts, A-D. Competing recombinant antigen was included as a control.

Day 3. Biosensor assay completed on ×15 plant extracts, 1-15 and ×4 CSSV negative plant extracts, A-D. Competing recombinant antigen at was included as a control.

Results and Discussion

Results of the competitive ELISA for CSSV are shown in Tables 9 and 10 (n=3). No plate reader was available at CRIG to measure absorbance, therefore the results were determined by eye by two people. Photographs are available and results will be analysed using image analysis.

TABLE 9Competitive ELISA results with a range ofCSSV positive and negative plant samplesSampleDescription ofNumberSample1 in 101 in 1001 in 10001New Juaben 1APPPNeg(Seedling)2NSABA (Mature plant,PPPNegyoung leaf)3KPEVEPPPNeg4KPEVEPNegNeg5New Juaben 1APNegNeg(Seedling)6New Juaben 1APNegNeg(Seedling)7New Juaben 1APNegNeg(Seedling)8New Juaben 1APPNegNeg(Mature plant,young leaf)9New Juaben 1APNegNeg(Mature plant,young leaf)10New Juaben 1APPPPNeg(Mature plant,young leaf)11New Juaben 1ANegNegNeg(Mature plant,red leaf)12KPEVEPNegNeg14NSABA (MatureNegNegNegplant, young leaf)15NSABA (MaturePNegNegplant, newflush leaf)16ANegNegNeg17B(P)(P)(P)18CNegNegNeg19D(P)NegNeg

TABLE 10Competitive ELISA results with 500 ngof recombinant antigen CSSV-CP-01Concentration (ng/ml)Sample4.51.510.30.10Recombinant antigenPPPPPPPPPNegKey: Negative Neg; Strong positives P, PP and PPP; Very weak positive (P)

The results show that 14 out of 15 plant leaf samples were positive for CSSV at a 1:10 to 1:100 dilution, no samples were positive for CSSV at 1:1000 dilution. The assay was able to detect CSSV infection by three strains of CSSV, New Juaben, KPEVE and NSABA. The Ghanaian ELISA assay only detects New Juaben. There were no convincing positives among the CSSV negative plant samples tested.

Subsequent laboratory QPCR results on the above samples have revealed that samples 1-15 contained CSSV. Melt analysis revealed two populations the New Juaben samples formed one group and the other strains formed another.

TABLE 11CSSV Biosensor results, n = 3 (technical)Sample123MeanSDSECVAntigen1570136015921507128428.4competitionNo competition151620052378196643214421.9Background100811031134108165216Sample 10118713051228124059194.8CSSV positive

The CSSV biosensor controls (italics) and a CSSV positive Sample 10. in Table 11 show that the Biosensor worked, and are illustrated inFIG.16.

A conceptual diagram of how the CSSV biosensor detection system will be used in the field is shown inFIG.17.

Example 2

Objectives:

Validate recombinant antibody Ab 31998.1 in the competitive ELISA with CSSV antigen 1 (CSSV-CP-01) with CSSV infected symptomatic, CSSV non-symptomatic and CSSV non-infected plant extracts.Compare competitive CSSV ELISA results with the number of CSSV DNA copies/plant cell present in each sample.Evaluate 3 antibodies: AbD31998.1, AbD31988.1 and AbD31999.1 in the CSSV competitive ELISA to CSSV-CP-01 antigen competition.Compare the binding of 15 recombinant monoclonal antibodies to CSSV antigen 1 (CSSV-CP-01) and CSSV antigen 2 (CSSV-CP-02).
Results:
CSSV Taqman QPCR

A good dose response was observed for 102-105copies of synthetic CSSV DNA (data not shown).

Summary of qPCR Data

CSSV infected plants—104to 105CSSV DNA copies detected.CSSV infected non-symptomatic plants—102to 103CSSV DNA copies detected.Uninfected plants—<50 CSSV DNA copies detected.

These data were normalised with plant DNA detected to determine the number of copies of CSSV DNA present per plant cell. This allows for differences in the success of DNA extraction to be accounted for.

Summary of qPCR Data of Plant Leaf Extracts from ‘CSSV Infected’Theombroma cacoaPlants

TABLE 12Symptomatic leaves contain 333.5-6.4 CSSV copies/cell; Non-symptomatic leaves contain 1.9-0.1 CSSV copies/cell.Plant G6Copies/cellPlant G46Copies/cellSymptomaticSample 257.9Sample 767.8Sample 266.4Sample 22333.5Sample 27254.6Sample 23100.2Sample 2816.6Sample 24298.8Non-symptomaticSample 201Sample 80.4Sample 300.2Sample 190.2Sample 320.1Sample 211.9Sample 320.4Sample 290.9
Summary of qPCR Data of Plant Leaf Extracts from ‘Uninfected’Theobroma cacoaPlants 0.3-0.1 CSSV Copies/Cell

TABLE 13Uninfected leaves contain 0.6-0.1 CSSV copies/cell (mean = 0.24)Copies/Copies/Copies/Plant AM2cellPlant AM4cellPlant AM6cellNegativeSample 110.2Sample 150.1Sample 330.1Sample 120.1Sample 150.2Sample 340.6Sample 130.3Sample 350.1Sample 140.2Sample 360.3Copies/Plant AM5cellSample 170.3Sample 180.2

Therefore from this experiment any sample containing >0.4 CSSV copies/cell is positive for CSSV.

The mean data for these values are displayed inFIG.18.

Validation of Reagents in the CSSV Competitive ELISA and Ability of the Assay to Detect CSSV in Plant Extracts.

FIGS.19A and19Brepresent summary data of the ELISA assays performed.FIG.19Ashows a dose response of CSSV recombinant coat protein.FIG.19Bshows the mean and SE of multiple leaves taken from the same plant.

Competitive ELISA using antibody AbD31998.1 shows results at three plant sample dilutions: 1/10 (FIG.20A); 1/100 (FIG.20B); and 1/1000 (FIG.20C).

CSSV competitive ELISA and Taqman qPCR results for individual infected CSSV plant extracts are shown inFIG.21. All samples with >45% inhibition in the CSSV competition ELISA had ≥0.4 CSSV DNA copies/cell. These samples are marked as positive for CSSV.

TABLE 14CSSV competitive ELISA and Taqman qPCR resultsfor individual non-infected plant extracts.SamplePlantDilution% inhibitionCopies/cell11AM2 neg A1/1042.60.21/10036.91/100031.412AM2 neg B1/1053.40.11/10030.21/100019.913AM2 neg C1/1034.30.31/100321/100019.37514AM2 neg D1/1033.10.21/10012.51/100019.515AM4 neg A1/10260.11/10010.41/10006.416AM4 neg B1/10400.21/10028.71/100023.717AM5 neg A1/1035.20.31/10022.51/100019.518AM5 neg B1/1044.10.21/10028.91/100028.533AM6 neg A1/105.80.11/10018.61/100011.134AM6 neg B1/1045.50.61/10016.21/10001035AM6 neg C1/1037.40.11/10019.81/100016.136AM6 neg D1/1036.10.31/10016.81/100010.8

2/12 CSSV non-infected plants had >45% inhibition in the ELISA, indicating some interference at the highest concentration tested (bold). However, one of those samples also had a qPCR result of ≥0.4 CSSV DNA copies/cell indicating a genuine CSSV positive.

Three anti-CSSV recombinant antibodies (Ab 31998.1, Ab 31988.1 and Ab 31999.1) were compared by competitive ELISA at 1.25 μg/ml of CSSV antigen 1 (CSSV-CP-01) (FIG.22). The observed antibody sensitivity to competition by CSSV-CP-01 was Ab31999>Ab31998>Ab 31988.

The interaction of the recombinant monoclonal antibodies with CSSV-CP-01 and CSSV-CP-02 was compared (FIG.23). Antibodies AbD31997.1 and AbD31998.1 interacted strongly with both antigens CSSV-CP-01 and CSSV-CP-02.

Summary of Results

Validate Recombinant Antibody Ab 31998.1 in the Competitive ELISA with CSSV Antigen 1 (CSSV-CP-01)

Using the Criteria of >45% Inhibition in the ELISA as being Positive for CSSV:11/12 symptomatic leaf samples were positive5/12 non symptomatic leaf samples were positive2/12 uninfected plant samples were positiveOnly 1 of the uninfected samples was confirmed as true CSSV positive i.e. >0.4 copies/cells by qPCR, therefore1 false positive was observedEvaluate and Compare 3 Antibodies AbD31998.1, AbD31988.1 and AbD31999.1Antibody sensitivity Ab31999>Ab31998>Ab31988Evaluate Binding of 15 Antibodies to CSSV Antigen 1 and CSSV Antigen 2 (CSSV-CP-02)All antibodies tested were originally selected for interaction with CSSV-CP-01. Antibodies AbD31997.1 and AbD31998.1 were found to have the highest binding with both antigens.Antibody AbD31998.1 has previously been shown to be the most sensitive to competition.

CONCLUSION

The Taqman qPCR has supported the results obtained in the CSSV competitive ELISA in that those plants with detectable levels of CSSV above 0.4 copies/cell were also positive in the competitive ELISA. For two out of three plants tested, CSSV was detected in symptomatic and >50% non-symptomatic leaves. These results were obtained with CSSV-CP-01 and antibody AbD31998.

Experiments have determined that antibody Ab31999 has greater sensitivity when tested with CSSV-CP-01.

Antigen CSSV-CP-02 was produced using sequences from currently circulating strains of CSSV in Ghana and the Cote D'Ivoire; the antibody AbD31998 is also the antibody that binds strongly to both antigens.

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