The Immunodiagnostics Group at the National
Diagnostics Centre has extensive expertise in all aspects of
antibody-based assay development, from immunogen preparation,
conjugation and antibody development to pilot-scale kit production
for market evaluation. The
group also have a track record in the development of cell-based
assay systems. Working
closely with other researchers in Ireland and overseas, they have
won national and international contracts for development of test
systems for targets of interest to the human clinical,
pharmaceutical, veterinary and food sectors.
The current focus of research in the
Immunodiagnostics group includes:
Ø Recombinant antibodies in
Rapid immunoassay technologies
Food-based assays including algal
Use of proteomics to identify new
Manager / Team Leader Immunodiagnostics,
University of Ireland,Galway.
353 91 524411 X 2071; 353 91 492071 (direct)
353 91 586570
Recombinant antibodies in
The development of good quality immunoassays
is entirely dependent on the availability of high performance
antibodies against the target analyte.
The Immunodiagnostics Group have, over many years, produced
several good antibodies, both polyclonal and monoclonal, for use in
assay development. More
recently with the help of a Marie Curie Development Host Fellowship
award from the EU, the techniques of phage display technology
have been established within the laboratory for the generation of
targeted single chain antibody gene libraries and the selection of
recombinant antibody fragments, scFvs, for use in assay development.
Single chain antibody libraries are being generated from
immunised mice, sheep, rabbits and chickens.
Working with a wide range of species is intended to maximise
the antibody repertoire available since the generation of
immunoglobulin diversity has taken quite different paths in even
closely-related species. Sheep, in particular, can develop a much
higher affinity immune response to many hapten targets of interest
than smaller animals; high affinity antibodies are essential for
sensitive assay development. Recombinant
antibody techniques are being used in the development of antibodies
against a wide range of target analytes, including specific algal
toxins, antibiotics and cardiac markers.
The group are also evaluating a range of approaches for the
improvement of antibody properties in vitro and the use of
the resulting scFvs in rapid immunoassay technologies
Rapid immunoassay technologies
The Immunodiagnostics group places a major
emphasis on the development of simple and convenient assay systems
without compromising assay sensitivity.
In collaboration with the Department of Biochemistry, NUI
Galway, they have developed highly sensitive microtitre
plate assays for direct determination of the concentration of
progesterone and estradiol in saliva.
The level of these steroids in saliva is approximately 1-2%
of that found in blood. Since
no extraction step is involved, assay time is less than 2h in both
There is now a growing demand for simple,
qualitative assay systems that give an answer in a matter of
Immunodiagnostics Group have developed a Latex Agglutination
assay for myoglobin, an early marker of cardiac damage.
Under their food assay programme in conjunction with
the National Food Centre, they have developed a Latex
Agglutination Inhibition assay for the detection of sulphonamide
residues in meat samples. They
have also developed Lateral Flow Membrane-based assays for a
range of analytes. Prepared
using the BioDot instrument, this assay format is the simplest yet
described, making them ideally suited for home, field or
More recently, the group in collaboration with
the National Centre for Biomedical Engineering Science at
NUI, Galway have initiated the development of automated
high-throughput assays for a range of analytes, utilising BIACORE™
biosensor technology. A
very simple, high throughput assay has been developed and validated
for the measurement of progesterone in raw milk samples, which has
potential for direct application in the milking parlour.
Until recently, the application of rapid
immunoassay technologies in food testing has lagged behind their
clinical applications. The
Immunodiagnostics group have, therefore, worked with scientists at
the National Food Centre in Dublin in the development of
user-friendly assay systems of relevance to food safety.
In collaboration with a local diagnostic company, convenient
microtitre plate and lateral flow membrane tests were developed for
the detection of E. coli O157:H7.
To assist in the control of Salmonella infection in pig
herds, an assay was developed for the detection of
Salmonella-specific antibodies in pork meat juices.
A broad-spectrum assay was developed for the detection of
mould contamination of cereals.
Assays have also been developed for the detection of
antibiotic residues in meat and meat products.
The Immunodiagnostics Group also work closely
with the Marine Institute in the development of assay systems
for the detection of algal toxins in shellfish.
The initial approach used was to develop and validate a cytotoxicity
assay because of the range of toxins that could be present in
the shellfish. By using
two different end-point determinations, the developed assay can
detect and differentiate both the diarrhetic shellfish poisons
(DSPs) and azaspiracid, a recently-discovered toxin.
These are the two major classes of toxin found in shellfish
from local waters.
A programme of work funded by the Advanced
Technology Research Programme of Enterprise Ireland has
now been initiated in collaboration with Dr. Gerard Wall,
Chemical & Environmental
Sciences Department,University of Limerick aimed at the development of a panel of
immunoassays for algal toxins, covering the increasing range
that are being found world-wide.
Both traditional and recombinant methods are being used for
antibody development and a range of assay formats will be developed.
Related work aimed at the detection of the toxin-producing
species is being carried out in collaboration with the Martin
Ryan Institute at NUI, Galway and funded by the Higher
Education Authority’s Programme for Research in Third-level
The NDC also has an interest in the
identification of food quality markers.
In this context, the Immunodiagnostics Group has recently
embarked on a research programme in collaboration with the National
Food Centre, aimed at the identification of markers of meat
tenderness and development and validation of convenient assays for
their detection. This is
also funded by the Advanced Technology Research Programme of Enterprise
The Immunodiagnostics Group have developed a
number of assays of clinical significance over the years, including
assays for the reproductive steroids, progesterone and estradiol
in saliva, and the cardiac markers, myoglobin and troponin T.
In collaboration with the Department of Biochemistry, they
have had a long interest in the development of convenient assays for
the detection of bone markers. Among
the assays developed and validated are a convenient
enzymeimmunoassay for the bone formation marker, osteocalcin,
and a HPLC assay for the collagen cross-link compounds, pyridinoline
and deoxypyridinoline. Monoclonal
antibodies have also been developed against cathepsin K, an
osteoclast-specific protease, that has potential for use as a marker
of bone resorption.
Immunodiagnostics Group have developed a number of assays for
veterinary applications. Their
equine fertility kit panel includes convenient microtitre plate
assays for determination of serum PMSG (pregnant mare serum
gonadotropin), estrone sulphate and progesterone levels.
Progesterone assays have also been developed and validated
for use with bovine serum and milk and there is an on-going
programme aimed at the development of an in-line biosensor for the
direct detection of steroids in cow's milk.
kit was developed for the estimation of total IgG content of bovine
serum, milk or whey protein concentrates (WPC) and several assays
have been developed for the detection of pathogen-specific
antibodies in bovine samples. The
feasibility of producing a whey protein concentrate with a high
titre against the gut pathogen, Helicobacter pylori, was
investigated in a recent research programme.
The resulting WPC was shown to be strongly bactericidal
against the pathogen, in vitro, suggesting a novel potential
application of these concentrates, which are an abundant by-product
of cheese production.
group are also working on a project, funded under the EU
INCO-Development programme and aimed at the development of
recombinant BCG multivaccines that protect against predominant
parasitic and epizootic diseases of ruminants in Latin America.
Their role in the project is to develop a cheap and effective
test to distinguish rBCG vaccination from natural infection.
group are also involved in an EU-funded project , Peptidex,
peptide binding specificities of fish major histocompatibility
complex (MHC) class I molecules.
The goal of this project, led by Dr. Iain Shaw (Iain.Shaw@nuigalway.ie),
is to develop a pathogen epitope prediction programme and evaluate
its usefulness in designing a viral pathogen peptide-vaccine.
The infectious salmon anemia virus (ISAV) will be used as a
model. This project will
develop much-needed reagents, such as antibodies and transfected
cell lines, for studying T-cell mediated immune responses in fish
and also for general fish research and will also provide a novel
approach to the development of vaccines for aquaculture.
Use of proteomics to identify new target analytes.
the modern world, there is an ever-increasing demand for improved
diagnostics to support the clinician in patient evaluation and
treatment. This requires
a greater understanding of the biological perturbations associated
with various diseases or drug treatments.
The Immunodiagnostics group have embarked on a programme, in
collaboration with the National Centre for Biomedical Engineering
Science at NUI, Galway, to apply the various 'proteomics'
techniques to the identification of novel potential target analytes
in selected disorders. The capillary electrophoresis and mass
spectrophotometric/data processing facilities recently established
at NUI Galway will form the backbone of this programme.
Howard, K., Kane, M.,
Madden, A., Gosling, J. P. and Fottrell, P. (1989) Direct
solid-phase enzymeimmunoassay of testosterone in saliva.
Clin. Chem. 35/10. 2044-2047.
O'Rorke, A., Kane, M.,
Gosling, J.P., Tallon, D.F. and Fottrell, P.F.
and validation of a monoclonal antibody enzyme immunoassay for
measuring progesterone in saliva.
Clin.Chem. 40/3, 454-458.
(1994) In Immunoassays: Laboratory
Analysis and Clinical Applications. Eds. Gosling, J.P. and Basso,
Boland, M.P., Sunderland,
D.H., Williams, D.H., Kane M., Headon, D.R., Roche, J.P.
(1994) Effect of immunisation of ewes against a1-26 inhibin
fragment on antibody titres, ovulation and lambing rate.
Animal Reproduction Science 34:241-251.
Lavin, F., Kane, M., Forde,
A., Shah, P., Gannon, F. and Daly, K. (1994) Comparison of five
cardiac markers in the detection of reperfusion following
thrombolysis in acute myocardial infarction. Br. Heart J.
O'Connor, A., Kane, M.,
Gosling, J.P. (1995).
The detection limit of radioimmunoassay as related to
antibody affinity. Biochem.
Soc. Trans.23(2) 393S
Forde, T. and Kane, M.
Diagnostic Systems for the Food Industry.
Dairy & Food, May, pp 2-3.
Tamate, K., Charleton, M.,
Gosling, J.P., Egan, D., Ishikawa, M., Fottrell, P.F., Kane, M.M.
colorimetric monoclonal antibody enzyme immunoassay for
estradiol-17b in saliva. Clin
O’Connor L., Joy J., Kane
M., Smith T., and Maher M. (2000)
Rapid PCR/DNA probe membrane-based colorimetric assay for the
detection of Listeria and Listeria monocytogenes in foods.
J Food Protect., 63: 337-342.
Kane MM and Banks JN. (2000)
'Raising antibodies’ in "Immunoassays, A Practical
Approach" Ed. James P. Gosling. Oxford University Press.Chapter
Kane M, Stevens FM, McCarthy
CF and Headon DR. (2000)
Effect of gluten-free diet on high density lipoprotein subfractions
in coeliac disease. Journal
of the Irish College of Physicians and Surgeons. 29 (1) 4-9.
O’Keeffe, M. J., Foran,
S., Kane, M. and O’Keeffe, M.
(2000) A rapid method for the on-site screening of pork
samples for sulphamethazine. Proceedings EuroResidue IV
Conference, Veldoven, The Netherlands, 8-10th May 2000, L. A.
van Ginkel, A. Ruiter (eds.) ISBN90-804925-1-5, 797-801.
Flanagan AF, Callanan KR,
Donlon J, Palmer R, Forde T, Kane M.
cytotoxicity assay for the detection and differentiation of two
families of shellfish toxins. Toxicon 39/7, 1021-1027
Early EM, Hardy H, Forde T,
Kane M. (2001)
Bactericidal effect of a whey protein concentrate with
anti-Helicobacter pylori activity.
J Applied Micro. 90(5) 741-748
Grogan, C., Raiteri, R., O’Connor, G.M., Glynn,
T.J., Cunningham, V., Kane, M., Charlton, M., Leech, D.
of an antibody coated microcantilever as a potential immuno-based
& Bioelectronics 17, 201-207.
Ishikawa M, Sengoku K, Tamate K, Takaoka Y, Kane M,
Fottrell PF. (2002)
The clinical usefulness of salivary progesterone measurement
for the evaluation of the corpus luteum function.
Gynecol Obstet Invest. 53(1):32-7.
Gillis E, Gosling J, Sreenan J, Kane M.
and validation of a biosensor-based immunoassay for progesterone in
bovine milk. J. Immunol
Methods. 267, 131-138.
McRedmond JP, Mulvihill NT, Kane M, Burke B, Aloul B,
Forde T, Walsh M, Fitzgerald DJ.
(2003) A rapid
latex bead agglutination assay to detect anti-streptokinase
Journal of Medical Science. Accepted.
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