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Panel of diagnostic reagents for immunofenotypisation in haematology and reumatology

The construction of new hybridoma cell lines producing monoclonal antibodies, construction of fluorochrome labelled antibodies for diagnostic purposes, and certification of selected reagents as in-vitro diagnostics.

The diagnosis and monitoring of malignant blood diseases and practically all rheumatic diseases now requires the effective integration of a wide range of diagnostic techniques and expertise. The cytomorphological aspects of blood cells and of their precursors both at the lymph node and bone marrow level are better defined and integrated by immunophenotypical investigations. This is a process of measuring the types of antigens expressed on cell surfaces by flow cytometry (FCM). Careful immunophenotyping by FCM facilitates accurate diagnosis in the leukaemia and lymphomas, and in some settings, may now also offer additional therapeutically relevant information. The same aims apply to the case of rheumatic diseases. A standardised nomenclature (CD, cluster of differentiation) is used internationally to categorise the monoclonal antibodies according to the antigen they detect. E.g. the characteristic immunophenotype of chronic lymphatic leukaemia (CLL), which represents 30% of reported cases of haematologic malignancies in the CZECH REPUBLIC, is: CD5+, CD23+, FMC7-, CD20 dim+, clonal surface immunoglobulin (sIg) dim+. But the immunophenotypic analysis by flow cytometry may also identify clinically relevant subsets of patients within CLL, e.g. CD38 positive immunophetype inform us on a poor prognosis of the examined CLL patients. In summary, careful immunophenotyping by flow cytometry facilitates accurate diagnosis of the chronic lymphatic leukaemia, other malignant lymphoma and rheumatic diseases, and may also offer additional therapeutically relevant information. The main advantage of the methodology is a synchronous application of two or more monoclonal antibodies against CDs and other antigens marked with various fluorochromes in one examined blood cell. Therefore the selection of monoclonal antibodies is the important first step for clinical use of FCM. The aim of the project is development of new monoclonal antibodies with different fluorochromes for immunophenotyping of blood diseases in haematology laboratory practice. The same antibodies will also be tested on the panel of patients with rheumatic diseases, with the aim being to increase the validity of laboratory testing. 1) Certification of selected reagents as In-Vitro Diagnostics (IVD), manufactured for commercial purposes. The main participant EXBIO PRAHA already possesses a large collection of monoclonal antibodies to CD antigens useful for immunophenotyping analysis. The panel of most commonly used antibodies labelled with PE and FITC is also available for research applications. However, for clinical diagnostic applications, these reagents should be classified as IVD (CE marked products) according to European Directive 98/79/EC. One of the most important parts of the certification process is the carefully planned and performed stability studies of the reagents that are pending IVD classification. The stability study will be carried out according to EN 13640:2002: Stability testing of in-vitro diagnostic reagents, which include the manufacture of three independent batches of the each reagent (PE and FITC labelled CD marker), and scheduled testing and data evaluation. The following panel of CD markers is selected: anti-CD3, CD4, CD8, CD14, CD16, CD19, CD20, CD45, CD56, HLA-DR. 2) Construction of new combinations of monoclonal antibodies (namely anti CD19, CD20, CD79b, FMC7, CD5, CD23, CD38, CD3, CD4, CD8) with different fluorochrome labelling for the accurate and fast immunophenotyping of chronic lymphatic leukaemia CLL. In order to determine more than two parameters, other flourochromes with emission spectra different to PE and FITC must be used for antibody labelling. Recently, a novel generation of fluorochromes called Alexa Fluor Dyes has emerged as having great potential in immunophenotyping by FCM. Alexa Fluor is the trademark of Molecular Probes (Oregon, U.S.A.) and the commercial use of these fluorochromes is available for licensing. The main focus will be on the preparation of a covalently coupled monoclonal antibody to various Alexa Fluor Phycobilliprotein TANDEMS and their thorough selection for immunophenotyping of CLL. Currently, there are only 6 companies, all located in U.S.A. that have the license for Alexa Fluor dyes. The aim of EXBIO PRAHA is to become the first European company to pioneer the use of these novel fluorochromes using a large panel of CLL patients and rheumatic diseases. The TANDEM currently available under licence from Molecular Probes would be: Alexa Fluor 610 R-Phycoerythrin, Alexa Fluor 647 R-Phycoerythrin, Alexa Fluor 680 R-Phycoerythrin and Alexa Fluor 750 R-Phycoerythrin. 3) Construction of hybridomas that produce monoclonal antibodies which specifically recognise the antigen ZAP-70 /syk tyrosine kinase which is a marker of antigen-mediated lymphocyte activation and poor prognosis for patients with CLL. EXBIO PRAHA is currently designing the expression of recombinant fragment of ZAP-70 in E.coli. The bacterially produced fragment will be used for immunisation of mice as well as for primary screening of antibody-secreting hybridomas. The suitability of a panel of ZAP-70 antibodies for intended clinical application will be screened on a panel of patients with CLL and rheumatic diseases. 4) Laboratory testing of fluorochrome-conjugated antibodies by FCM and confirmation of the results with microscopical morphology in 250 patients with CLL before the recommendation for their diagnostic use in haematology practice, and 150 patients with RA (Rheumatic Arthritis), SLE (Systematic Lupus Erythematosis) and other patients with rheumatic diseases. Keywords: monoclonal antibodies, clinical studies, haematology.
Acronym: 
HEMATU
Project ID: 
3 389
Start date: 
01-01-2005
Project Duration: 
36months
Project costs: 
370 000.00€
Technological Area: 
Diagnostics, Diagnosis
Market Area: 

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