Portable immunodiagnostics for on-farm
parasitic disease monitoring
Eithne Dempsey1, Brian Seddon1, Baljit Singh1, Simon Egan1, James Hayes1, Mary Sekiya2, Grace Mulcahy2
1
Microsensors for Clinical Research and Analysis, ITT Dublin, Tallaght, Dublin 24, Ireland,
2 School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
On-the-spot testing technology is yet unrealised for disease control on farms. The remit of ImmunoTouch is to
develop, field-test and validate a mobile electronic system for early diagnosis of parasitic/infectious diseases onfarm. ImmunoTouch is composed of a handheld electronic reader and sample assay card. A blood or bulk tank milk
sample is applied to the card, which contains various reagents required for a specific ELISA procedure. Once the
card is inserted into the reader the test begins. The project focuses on Fasciola hepatica infection as a model
disease to assess the ImmunoTouch technology in field use. The device was also tested with other animal parasite
diseases such as Ostertagia ostertagi (stomach worm) and several targets in human diagnostics. Test run times are
about 10 minutes for a quantitative result compared to 150 min for a standard laboratory ELISA.
1) F. hepatica stained in cross section within the host
liver bile duct. 2 & 3) Adult liver fluke.
Life cycle of liver fluke
Liver fluke disease or fasciolosis is an infestation of ruminants caused by the common trematode parasite,
Fasciola hepatica (temperate) and Fasciola gigantica (tropical climates). Fasciolosis is found throughout the
world and has a major impact on livestock productivity, particularly beef and dairy. Depending of the severity of
infection liver fluke can cause significant disruption in bovine milk production (≤ 8%) and a loss in meat
production (≤ 20%) in cattle and 30% in sheep. It is estimated that fasciolosis causes an annual loss of ~€2.5
billion worldwide, and a loss to the Irish industry in the region of ~€90 million. Adult fluke reside in the bile
ducts of host animals, where eggs are shed in faeces onto grassland. Here the eggs hatch into mobile
miracidia, which have a short life-span and must come into contact with their intermediate host (freshwater snail
in the Lymnaeidae family) within hours. After a snail is infected, development of the miracidia continues to the
final snail stage, the cercaria. These mobile cercarias are shed by the snail and attach themselves to blades
of grass where they encyst to form metacercariae. These metacercariae are then ingested by the final host (e.g.
cow or sheep) and migrate from the small intestine through the gut wall to penetrate the liver.
Areas endemic for fasciolosis
(marked in red)
Reagent System
The ImmunoTouch assay card consists of four
sealed cavities to store micro-litre volumes of
ELISA reagents. Right: tear-drop design of a
reagent cavity. This geometry allows hydraulic
focusing of solution towards a pressure-sensitive 4
valve. Actuation pins in the card reader force
sample and reagents3 throughout a channel
network to an immunoelectrode detector or
reaction chamber. Below: ELISA reagent stability
studies for the fluke assay. Considered here is the
long term stability of peroxidase conjugate and
substrate formulation.
Sampling
Electronic Reader
ImmunoTouch utilises a removable strip
technology with capillary flow fluidics for milk or
blood collection. The strip has a filtration
function to remove cells and fat from a sample.
Once inserted into the card and the card placed
in the electronic reader, the membrane touch
actuation releases a metered volume to the
immunoreactor or detector.
A handheld card reader of the ImmunoTouch
assay
system
performs
sequential
electromechanical actuation for reagent
delivery and micropotentiostat control for
electrochemical data acquisition.
Fluidics
Pressure-sensitive cavities are housed within
the ImmunoTouch card. Reagents move by a
consecutive process on actuation of membrane
touch fluidics for release to a detector.
Poiseuille equation for laminar liquid flows in
rectangular micro-channels allows calculations
on volume & linear flow rates and reagent
capacities related to hydraulic force-pressure
gradients and micro-channel dimensions.
5
Top left, ImmunoTouch card and electronic
reader. Right, design and test unit for
electromechanical pin actuation. Below,
prototype electronic reader design layout and
display functions.
Conjugate and TMB time course experiment over an 11 week period. All reagent formulations for the liver fluke ELISA,
enzyme substrate TMB/H2O2 and peroxidase conjugate are stable at 25ºC over a one-month period. This degree of stability
is sufficient for the current stage of development of the ImmunoTouch ELISA card. Absorbance was measured at 450 nm.
U/A > 10mm/s, V = 10-100µL.
.
Detection
The ImmunoTouch detector is a series of electrodes coated with
antigens specific for the target antibody, i.e. cathepsin L1 (CL1)
proteinase from liver fluke. The detector measures peroxidase
levels in a thin-layer flow cell.
Prototyping
Peroxidase Electroanalysis
CL1
ImmunoTouch assay card is a credit-card size device for mobile ELISA applications. It is constructed
from film plastics and laminates and precision machined by laser cutting and etching. The detection
system is composed of a series of screen printed electrodes modified with immunochemical reagents.
1
2
BSA
Illustrated above is the immunoelectrode flow cell (left) used in the
ImmunoTouch detector. Centre is the ELISA protocol showing the
capture of a fluke antibody and peroxidase sensing. Right is a
typical coulometry signal of the CL1 immunoelectrode and a BSA
electrode control for the fluke immunoassay.
Electrode Design
ImmunoTouch detection methodology is based on charge
measurement or coulometry for the detection and the sensitive
quantitation of peroxidase labels in ELISAs.
The detector operates by applying an excitation potential
difference across sets of electrodes followed by charge
measurement with a microcoulometer. The detector can operate
with on-electrode capture molecules such as antibodies or
antigens - immunoelectrode detector, or with off-electrode capture
molecules - immunoreactors. The latter requires additional microfluidics integrated to the ImmunoTouch detector. In this case the
detector effectively remains isolated from the ELISA reagent
system.
Above left: peroxidase substrates screened for
application to the ImmunoTouch detector.
Right: Electrochemistry of TMB.
ABTS
3,3',5,5'-tetramethylbiphenyl-4,4'-diamine
ABTS and TMB substrates are used in
peroxidase immunoassays. TMB is the reagent
selected for fluke assay system. ABTS has
been utilised in Ostertagi ostertagia tests.
Out of Laboratory Applications
• Human IVD
• Livestock infectious
diseases
• Food allergen and security
testing
• Faecal pathogens
• Pyrogens
On-card ELISA with ImmunoTouch was demonstrated by loading cavities with fluke
assay reagents (sample, wash, peroxidase conjugate and enzyme substrate). Fluke
assays were run in 15 minutes. Detection response to fluke antibodies in milk samples
is shown in the chronocoulometry trace (right). The study investigated standard
method correlations, assay time parameters and card inter-card variations.
Institute of Technology Tallaght , Dublin 24, Ireland.
Tel: +353 1 4042862; Fax +353 1 4042404
Email: [email protected]
CL1
2
ITT Electrochemical Response x 10 /nC
BSA
3
ImmunoTouch has established
laser machine processing to
precision engineer and prototype
the main components of the
membrane touch fluidic system.
The R&D has focused on an
integrated approach to screen print
detector design and
laser
machined film plastics for rapid
prototype design and testing.
The scheme (right) shows some of the
major evolution stages of the
ImmunoTouch card design.
1. Linear flow-through micro-channels
connecting
membrane
touch
reagent cavities to a sink.
2. Application of a printed electrode
detector.
3. An early device with sampling
component using syringe injection.
4. Pinnate channel network for
efficient consecutive flow fluidics.
5. Introduction of multiplex detection
capabilities and an independent
strip sampling device.
Proof-of-Principle
Contact details
Prof. Eithne Dempsey
MiCRA-Biodiagnostics
1. Membrane touch
Fluidics.
TMB
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
Correlation – milk testing
The ImmunoTouch assay data is correlated with the UCD ELISA optical measurement
(O.D. – 450nm absorbance) using milk samples with widely varying fluke antibody
levels (UCD ELISA S/P score, 0 – 152). The study reveals that the electrochemical
detection methodology has a linear correlation (r2 = 0.9943, n=26) with the standard
optical method. Precision with ImmunoTouch is relatively better for mid to high range
compared to low fluke antibody levels. This promising result indicates that the device is
a reliable indicator of low and high fluke antibody levels in milk, and can be applied to
in-field testing.
2. Integration of fluidics
components with a printed
electrode detector.
3. Injection sampling
Components.
4
4. Pinnate channel
network with pop
valves.
5
5. Advanced ImmunoTouch card
with multiplex detection system,
membrane touch fluidics and
sample injection strip.
18
16
2
R = 0.9943
14
12
10
8
6
4
2
0
0.0
0.5
1.0
1.5
UCD Optical Response (O.D.)
2.0
Conclusion
ImmunoTouch has advanced immunoelectrode detection methodology including a multiplexed testing
functionality and established proprietary “membrane touch” mesofluidics (European Patent Application
No. 13182873.3) with novel sample interface components. ImmunoTouch R&D has built and tested a
custom-designed electronic reader unit and software for fluidics actuation, detector control and data
acquisition. Of the 26 milk samples in a correlation study, 24 (92.3%) matched the standard ELISA
assignment. The predictive outcome for the ImmunoTouch system – i.e. capability of registering a
correct assignment (fluke status) for a sample was established in nine out of ten tests - 90% (one false
positive, no false negatives).