in field detection of artemisinin · 2010. 4. 6. · victoria lawson sensapharm ltd. overview •...
TRANSCRIPT
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Development of antibodybased detection systems for Artemisinin
WHO/MMV Artemisinin Conference 2009
Victoria LawsonSensaPharm Ltd
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Overview• Why? • Making antibodies to Artemisinin• Development of Artemisinin ELISA• ELISA testing• ELISA kit• Artemisinin LFD development• LFD testing and optimisation• Artemisinin LFD kit• Future work
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Background
• Need for a simple, cheap, robust test for Artemisinin in dry/wet leaf
• Laboratory and in field use• Review existing technologies• Select technology (LFD)• Develop test system (ELISA)• Field test device/ customer feedback• Launch test
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Antibody productionStep 1: Conjugation• Artemisinin too small (hapten) to cause an immunological response, must be
coupled to carrier protein e.g. BSA, ovalbumin• Two different methods used:1. Artemisinin – No reactive groups Mannich reaction2. Artesunate – Reactive carboxyl group EDC conjugation
Step 2: Immunisation• Conjugates injected into New Zealand female rabbits over period of 8 weeks –
standard protocol
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Preliminary ELISA tests• Antibodies to be used in tests affinity purified using protein A and quantified• Reactivity with ASOVA tested by direct ELISA (determine titre)
• All antibodies reacting well Try competitive ELISA
Principle
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Competitive Artemisinin ELISA• Test reactivity of affinity purified antibodies with artemisinin and produce
standard curves for each antibody• Artemisinin in solution will bind with antibody, reducing amount of free
antibody left to bind with the ASOVA coated plate, the signal will therefore reduce as the artemisinin concentration increases
• Working assay for pure artemisinin crystals in solution, test on A. annua extracts
• Limits of detection = 1mg/ml50ng/ml
• Limits of quantification = 264µg/ml
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Testing ELISA on Extracts• Quantification of artemisinin using ELISA was tested using 2 different antibodies
for comparison
• The antibodies tested showed good agreement in artemisinin quantification
• Compare ELISA to other analytical techniques
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Comparison of ELISA to other techniques
• Accuracy of artemisinin quantification by ELISA was tested by comparing to other used techniques
• ELISA proves to be as consistent as and comparable to other techniques, giving similar quantifications in extracts
• We can confidently move to LFD development using tested antibodies
• Other antibodies ready for use
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Antibody Cross reactivity• Test reactivity of antibodies with range of artemisinin related compounds
expected to be present in A. annua extracts• Test works on same principle as competitive ELISA, the lower the signal, the
higher the reactivity with the antibody
• Observed cross reactivity much lower than those previously reported
• All antibodies react with artemisinin much more than any related compounds0.5
0.6
0.7
0.8
0.9
1
1.1
0 50 100 150 200 250
Concentration (ug/ml)
A/A
o (4
50nm
)
AM (100%)
DAM (25.8%)
AT (24.2%)
AA (21.5%)
DHA (20.3%)
DHAA (26.8%)
AS (24.4%)
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ELISA testing The artemisinin ELISA has been extensively tested with respect to:
•Inter and intra assay reproducibility CV=0.04
•Dilution parallelism mean recovery = 99.7%
•Spike recovery overall mean recovery = 100.5% (96.4107.6)
•Sample stability and storage
•Specificity
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ELISA kit• To provide quick and accurate analysis of artemisinin in extracts
as alternative to LCMSMS and HPLCUV, TLC etc• For use with A. annua extracts, pure or partially purified drugs
and clinical samples• Complete and ready to use• Colorimetric detection• Detection of artemisinin between 1mg/ml and 50ng/ml• Quantification of artemisinin between 2 – 64µg/ml
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Lateral Flow Strip Development• Polyclonal antibody conjugated to blue latex microspheres• ArtesunateOVA conjugate sprayed onto various nitrocellulose membranes
(Test line)• Secondary anti rabbit antibodies also sprayed onto membranes (Control line)
5mm from test line• Interaction of antibody conjugate with C and T lines analysed in initial proof of
principle test
1 H
F 18
0
2 H
F 13
5
3 C
N14
0
4 C
N95
5 P
rim
a 85
6 P
rim
a 40
• Showed positive interaction on strip
Investigate inhibition of T line by artemisinin
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LFD Competitive Test• Artemisinin in the extracts tested will compete with the test line to bind to the
antibody conjugates, as the artemisinin in the extract increases, the test line will decrease in intensity
• Initial tests showed progressive inhibition of T line with increase in artemisinin concentration
• Artemisinin extracted from 0.1g A. annua using bicarbonate buffer
Neg
ativ
e
1/64
dilu
tion
1/32
dilu
tion
1/16
dilu
tion
1/8
dilu
tion
1/4
dilu
tion
1/2
dilu
tion
• Upon initial positive demonstration of competitive assay, second round of assay development performed
• Different component materials tested for optimisation e.g. membranes, C and T line concentrations
•Two prototypes produced
•Incorporated into assembled LFD cassette for initial dry testing
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Dry Assay Tests• Testing of LFD gave visible results showing
inhibition of T line with addition of artemisinin extract from A. Annua
• Working assay using LFD try quantification using reader
• Measurement of Tline intensity by LFDR101 reader showed a direct correlation between Artemisinin concentration and Tline intensity
AM conc. (µg/ml) LFD 1 LFD 2 LFD 3 Mean
Standard deviation
2.66 79.4 80.6 79.6 79.8 0.6431.33 84.2 84.3 83.6 84 0.3790.67 84.8 83.2 84 84 0.8000.33 80.4 78.8 79.6 79.6 0.8000.17 75 74.6 74.8 74.8 0.2000.08 70.9 70.6 69.5 70.3 0.7370.04 66.1 67.4 66.9 66.8 0.6560.02 64.4 65.4 66.6 65.4 1.102
Negative 61.2 63.6 61.7 62.1 1.266
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Quantifying extracts using LFD• Measurement of Tline intensity by LFDR101 reader showed a direct correlation
between artemisinin concentration and Tline intensity
• Measures artemisinin between 0.03 and 0.6µg/ml• Quantifies between 0.22.5% w/w artemisinin
Sample DFT-line value
conc. (µg/ml) Ratio
%w/w AM
Leaf 1 40 741 17.2 9.7 0.83Leaf 2 40 929 6.3 19.5 0.61Leaf 3 40 741 17.2 12.2 1.05Leaf 4 50 887 9.9 25.7 1.26Leaf 5 40 683 23.5 7.4 0.87
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In Field Extraction Kit• For use with LFD and reader in field• Short extraction time (60s) removing just trichomes• All equipment included
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Future Work
• ELISA – offsite testing, production of kit for distribution
• Artemisinin LFDs – Batch testing, quality checks• LFD Reader – software final calibration and
validation• Production of full LFD/Reader kit for distribution• Pharmaceutical testing – test ELISA/ LFD in
comparison to current colorimetric method
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Acknowledgements• Medicines for Malaria Venture• Complement Genomics Ltd• Botanical Developments Ltd• Forsite Diagnostics• East African Botanicals• Mediplant• Biogenes• FSC Developments Ltd• DeMontfort University• University of Bath
•KamTech Ltd•High Force Research Ltd
Development of antibody-based detection systems for ArtemisininOverviewBackgroundAntibody productionPreliminary ELISA testsCompetitive Artemisinin ELISATesting ELISA on ExtractsComparison of ELISA to other techniquesAntibody Cross reactivityELISA testing ELISA kitLateral Flow Strip DevelopmentLFD Competitive TestDry Assay TestsQuantifying extracts using LFDIn Field Extraction KitFuture WorkAcknowledgements