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 diagnostic

Ø  Rapid immunoassay technologies

Ø  Food-based assays including algal toxin detection

Ø  Clinically-relevant assays

Ø  Veterinary assays

Ø  Peptide vaccines

Ø  Use of proteomics to identify new target analytes

Contact:

Dr. Marian Kane,

Centre Manager / Team Leader Immunodiagnostics,

National Diagnostics Centre,

National University of Ireland,Galway.

Ph: 353 91 524411 X 2071; 353 91 492071 (direct)  

Fax: 353 91 586570

Email:   Marian.Kane@nuigalway.ie

Ø             Recombinant antibodies in diagnostics

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

Ø             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 cases. 

There is now a growing demand for simple, qualitative assay systems that give an answer in a matter of minutes.  The 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 point-of-care use.

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.

Ø             Food Assays

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 Institutions (PRTLI)..

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 Ireland.

Ø             Clinically-relevant assays

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.

Ø             Veterinary assays

The 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. 

A 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.

The 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.

Ø              Peptide vaccines

The Immunodiagnostics group are also involved in an EU-funded project , Peptidex, studying the 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.

Ø             Use of proteomics to identify new target analytes.

In 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.

Publications

§         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.  (1994).   Development and validation of a monoclonal antibody enzyme immunoassay for measuring progesterone in saliva.  Clin.Chem. 40/3, 454-458.

§         Kane, M.  Cardiac  markers. (1994) In Immunoassays:  Laboratory Analysis and Clinical Applications. Eds. Gosling, J.P. and Basso, B.V.  Butterworth-Heinemann, pp. 235-247.

§         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.   73: 422-427.

§         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.  (1996)  Emerging 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. (1997)  Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17b in saliva.  Clin Chem 43:7,1159-1164.

§         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 2, pp19-58

§         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.  (2001)  A 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.  (2002)  Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor.  Biosensors & 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.  (2002)  Development 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 antibodies.  Irish Journal of Medical Science.  Accepted.

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