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Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes Niamh Gilmartin Dublin City University
Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes
Niamh GilmartinDublin City University
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88 acres of campus; 11.5K+ students.Degree programmes in Analytical Science, Biotechnology and Biomedical
DiagnosticsNational Centres of Excellence (NCSR, CBAS, BDI).Dedicated Innovation, IP and Commercialisation centre.
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• Principal investigator • Professor Richard O’ Kennedy.• 1 research fellow• 6 postdoctoral researchers.• 12 postgraduate researchers.• 6 technical staff.• Over 482 research publications to date, 60 PhDs and 30
MSc. from the group to date
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Biorecognition molecule:Antibodies, fragments, peptides,protein scaffolds and DNA
Antibody Production:Monoclonal, polyclonal and recombinant (human, chicken, mouse and rabbit)
Fermentation:Large scale protein expression
Cloning:Cloning, expression and purification of antigens and biomarkers
Protein Kinetics:Determination of interaction rate constants and thermodynamicprofiles
Liposome's:Antibody labelling anddye/contrast agent
encapsulation
Automated Screening:Custom written software for high throughput screening
Mutagenesis:Random and site-specificmutagenesis for proteinimprovement
Display and Selection:Phage, yeast and ribosomal display of proteins
Lateral Flow Assays:Point of care tests for environmental and clinical applications
Biosensor Assays:Incorporation of biorecognition elements into biosensor platforms
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Marine ToxinsProstate Cancer
Markers
Aflatoxins
Food StuffContamination
Anti-protozoan and Anti-parasitic drugs
Cardiac Markers
Multiple Myeloma
Sialic Acids
Foodborne bacteria
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• Generation of a range of antibodies and antibody fragmentsto haptens, proteins, whole cells and bacteria.
• Development of rapid immunoassays and biosensor assaysfor environmental, food and clinical analysis.
• Engineering of antibodies for improved assay sensitivity andantibody stability.
• Development of high throughput protocols for antibodyscreening and characterisation.
Essential in antigen binding
• A single antibody binds two antigens, • Both these two antigens are bound to the antibody at the same point on the antigen
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Depends on the antigen and the assayPolyclonal AntibodiesRelatively quick and inexpensive to produceCapable of recognising multiple epitopes on any one antigen and therefore they:◦ Can help to increase the signal produced by the target protein as the antibody
will bind to more than one epitope◦ Less sensitive to antigen changes than monoclonal antibodies◦ Useful when the nature of the antigen is unknown◦ More robust detection due to multiple epitopes
Monoclonal AntibodiesUnlimited quantities of highly specific antibodiesAll batches will be identical and specific to just one epitope The high specificity of monoclonal antibodies decreases background noise and cross-reactivity,
helps provide reproducible results
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Requirements• Source of Target• Target specific antibody
(i.e. monoclonal, polyclonal or recombinant)• Convenient assay format
(e.g. sandwich immunoassay)• Reliable calibration procedure
Ideal Properties• Specificity• Sensitivity• Wide measuring range• Robust• Low false positives/negatives• High measuring accuracy and
precision• Amiable to measurements in crude
samples• Simple methodology• Inexpensive• Rapid
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• Complexity of sample to be tested i.e. food particles, other contaminants
• Generation of bacteria specific antigen• Generation of an antibody against a pathogenic strain of
the bacteria• Ensuring that the antibody will bind to cells in conditions
such as biofilms
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Pathogen Technique Detection method Detectionlimit CFU/mL
S. typhimurium FLISA (Quantum dots) Fluorescence 1.95 x 103
Copper enhanced gold nanoparticles Electrochemical 98.9
FLISA (magnetic nanoparticle + quantum dots)
Fluorescence 103
Localised SPR Optical 102
Camplyobacter Voltometry with multi-walled carbon nanotubes
Electrochemical 400
Indirect ELISA Optical 104-106
L. monocytogenes ELISA Optical 1-10Surface plasmon resonance Optical 105
E. coli O157:H7 Flow cytometer (ruβpy-doped silica NPs) Fluorescence 1FLISA (magnetic nanoparticle + quantum dots)
Fluorescence 103
Surface plasmon resonance Optical 107
Piezoelectric-excited millimeter-sized cantilever
Electrochemical 1
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• Generation and purification of antibody against L. monocytogenes
• Development of immunoassay for the capture and detection• Integration of the immunoassay into the sampling and
detection systems developed by the other partners• Large-scale production of antibodies for trials
Internalin A (InlA)80 kDa
Internalin B (InlB)~65 kDa
P66 (kDa)Aminopeptidase
Listeriamonocytogenes
Anti-InlA monoclonal antibody 2B3 (mAb 2B3)
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A) Competitive ELISA showing the specificity of the antibody. B) ELISA showing mAb 2B3 binds different serotypes of L. monocytogenes.
1
3
5
7
9
11
13
15
A/A
o
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1a 1/2a 1/2b 1/2c 3a 4bAb
s @
450
nm
L. monocytogenes serotype
A B
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Detection antibody
Capture antibody
Internalin A (InlA)80 kDa
Listeriamonocytogenes
Anti-InlA monoclonal antibody 2B3 (mAb 2B3)
magnetic particle
Fluorescent label
mAb 2B3
Antibody purification
Conjugation to magnetic particles
Fluorescent labelling of antibodies
Optimisation of capture of L. monocytogenes using
magnetic particles
Optimisation of FLISA for the detection of L.
monocytogenes
Immunoassay for the capture and detection of L. monocytogenes
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0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
1,00E+001,00E+011,00E+021,00E+031,00E+041,00E+051,00E+06
FU e
x 40
5nm
/ em
655
nm
L. monocytogenes CFU/mL
Limit of detection (<1 x 102 CFU/ml)
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Culture of mAb 2B3 hydridoma cells in Dulbecco’s modified Eagle’s medium.
PurificationProduction Labelling
Before BIOLISME◦ Assay carried out in lab◦ Pre-concentration of L.
monocytogenes samples required (can take up to 3 days)◦ Immunoassay takes 3-4 hours and
requires 37oC incubation◦ Could detect 105 cfu/ml ◦ Time consuming, laborious and
requires expert personal
As a result of BIOLISME◦ On-site assays and results◦ Rapid pre-concentration done
on site using antibody coated magnetic particles
◦ Immunoassay takes <30mins and can be done at RT◦ Can detect <100 cfu/ml◦ Rapid, easy to use and user
friendly◦ Can detect L. monocytogenes in
biofilms◦ Can detect only live cells
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• Easy to use capture and detection system for L. monocytogenes which can detect below 100 cfu/ml
• Results in: • L. monocytogenes specific detection system• Reduction in waiting time for lab results• Increased confidence in cleaning regimes• Assays can be done on-site
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