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Design, production and testing of monoclonal antibodies against BTK for flow cytometric aplication

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Design, production and testing of monoclonal antibodies against BTK for flow cytometric aplication

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Bruton's Tyrosine kinase (BTK) is a protein that has important functions in signaling pathways downstream of the B-cell antigen receptor. A mutation in the Btk gene results in the disease X-linked agammaglobulimia (XLA). XLA is a primary immunodeficiency, a congenital disorder of the immune system. Patients diagnosed with XLA have a block in their B-cell differentiation, which results in almost no mature B cells and low antibody titers. These patients suffer from recurrent bacterial infections and need life-long immunoglobulin replacement therapy. This report describes the design, production and testing of monoclonal antibodies against BTK.

Early diagnosis of XLA is of vital importance; the chance of bacterial infections will be reduced when therapy is given as soon as possible. At the moment, molecular techniques are used for the diagnosis of XLA. The exons of the Btk gene are sequenced to determine the mutation, which takes 2-3 days and is relatively expensive. When monoclonal antibodies against BTK are available, patients can be diagnosed by flow cytometry within 2-3 hours. For this purpose, the presence of BTK is made visible using intercellular staining with a monoclonal antibody against BTK. However, such a monoclonal antibody does not exist yet.

The production of a monoclonal antibody involves a number of steps. The first step is to decide which fragment of the protein the antibody must recognize. This protein fragment should not cross react with any other human protein. The protein fragment, within the natively folded protein, should be accessible for the antibody to bind. Two protein fragments were chosen for the immunization of mice, both at the N-terminus of BTK, where little cross reactivity with other proteins exists. The chance of an immune response of the mice against one of protein fragments is higher when multiple protein fragments are chosen.

To produce the BTK DNA fragments by PCR, two primer sets were developed. The PCR products were ligated into prokaryotic expression vectors. The generated plasmids were transformed into E.coli BL21 competent cells for protein production. The purified protein fragments were checked by Western blot; protein bands around the expected molecular weight were observed. Four mice were immunized with 28 mg of BTK protein fragment 1 and four mice were immunized with 32 mg BTK protein fragment 2. This is the stage in which the project is at this moment. 56 days after immunization, blood samples of the mice will be taken, to determine whether the produced antibodies are directed against the protein fragments of BTK in its native folding. For this purpose, COS-cells expressing the full-length BTK were generated and tested using the commercially available polyclonal antibody a-BTK C20. For each BTK protein fragment, the mouse with the highest titer will be selected. The mice will be sacrificed and spleen cells will be fused with SP2/0 tumor cells to obtain antibody-producing hybridomas. 2-3 weeks after fusion, positive clones will be selected by COS-cell screening and will be subcloned.

4-5 weeks after subcloning, several other tests will be used to select the most optimal hybridoma clones. In this report, these tests were performed using hybridomas and antibodies of other project. Supernatants of hybridomas will be tested by ELISA, to determine the isotypes of the antibodies. Hybridoma cells will be tested by Gene Scan analysis to confirm that the hybridomas are unique and monoclonal. If possible, three different hybrimomas will be selected and grown in large culture. When the hybridomas are cultured for 2-3 weeks, supernatant will be harvested and IgG antibodies will be purified. The antibodies will be tested by Western blot and in flow cytometry, both on cell lines and patient samples. The monoclonal a-BTK antibody that has the highest affinity and that works best in all assays will be used for diagnosis of XLA-patients.

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OrganisatieHZ University of Applied Sciences
OpleidingChemie
InstituutAcademie voor Technologie & Innovatie
PartnersErasmus MC, department of Immunology, Rotterdam
Gepubliceerd in
Datum2006-06-29
TypeBachelorscriptie
TaalEngels

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