Interest in any of the products, request or order them at Bio-Connect Diagnostics.

Transcription

1 CE IVD Minicatalog Interest in any of the products, request or order them at Bio-Connect Diagnostics. Bio-Connect Diagnostics B.V. T NL +31 (0) T BE +32 (0) Begonialaan 3a F NL +31 (0) F BE +32 (0) TE Huissen E info@bio-connectdiagnostics.nl The Netherlands W

2 In Vitro Diagnostic Products Immunology pp pp pp Immunophenotyping Allergy Phagocytosis

3 EXBIO Praha Dedicated to Superior Cytometry Reagents Mission EXBIO Praha strives to exceed the most demanding customer expectations in the field of analytical cytometry by providing a comprehensive range of high quality products and services at affordable prices. EXBIO Praha is a dynamic privately-owned European biotechnology company focused on design, development, manufacture and sale of monoclonal antibodies and other reagents for research and clinical applications. Who we are EXBIO Praha was established in 1998 and soon became a trusted and reliable OEM supplier to all leading antibody vendors. Our flow cytometry antibodies have become widely recognized reagents among life science researchers and diagnostic laboratories. In addition to OEM supplies, we distribute our products worldwide under the EXBIO trademark through a network of experienced local distributors. 2

4 Our portfolio Flow cytometry validated antibodies for a broad range of research applications (immunology, hematology, cancer research, etc.) available in many colors and formulations (suitable for 8-color immunophenotyping). Pre-mixed antibody conjugate cocktails for in vitro diagnostic (CE IVD) applications (immunophenotyping of white blood cells). Flow cytometry assays (FlowEx Kits) for comprehensive diagnosis and analysis of patient health status: FagoFlowEx Kit for examination of phagocytosis (CE IVD), BasoFlowEx Kit for analysis of allergy specificity (CE IVD), ApoFlowEx FITC Kit for detection of apoptosis (RUO) and SpermFlow Kit for evaluation of human semen quality (RUO). Other monoclonal antibodies for applications such as Western blotting, immunohistochemistry, immunoprecipitation, ELISA or functional in vivo studies. Antibodies to cytokeratins, betaiii-tubulin, shla-g and IgE belong to our best sellers. shla-g ELISA kit for determination of soluble forms of Human Leukocyte Antigen-G (shla-g) in amniotic fluid, cell culture supernatant, plasma and serum. EXBIO products are offered under the following brands: Reagents and kits for flow cytometrybased in vitro diagnostic applications: Immunophenotyping Examination of allergy Examination of phagocytosis Monoclonal and polyclonal antibodies and kits for research applications. 3

5 Facilities and Perspectives In 2003 we opened an 800 m 2 manufacturing plant in Vestec near Prague (EXBIO I). As the company was growing dynamically, construction of much larger building had to be started soon. In 2010 we opened a brand new m 2 Research and Technology Centre EXBIO in Vestec (EXBIO II), which significantly enlarged our manufacturing capacity, including up-stream processes in large-scale bioreactors designed for perfusion cell culture in serum-free medium (single antibody batch up to 10 g), purification steps (low-endotoxin antibodies) or antibody conjugation procedures. The heart of this building is the core flow cytometry facility In 2014 the opening of an additional m 2 manufacturing plant unit (EXBIO III) is scheduled to provide us with further manufacture equipment and facilities. We plan to continue strengthening our manufacturing capacity (single antibody batch up to 100 g) in the following years, and we are continually optimizing the manufacturing process regarding highly reliable automatization and effectivity. We can accomodate the growing demands for contract antibody manufacturing services. 4

6 Quality statement EXBIO Praha, a.s. is dedicated to its customers and to providing them with the products and services that are of the highest quality and that facilitate successful research. Our company has been assessed and certified as meeting the requirements of ISO 9001:2008, ISO 14001:2004, and ISO 13485:2003 for the following activities: Design, development and production of biotechnological products for research use. Design, development and production of in vitro diagnostic class D. Regulatory Notes reagents are intended for in vitro diagnostic use in laboratories outside USA and Canada. These reagents conform to the European In Vitro Diagnostic Medical Device Directive 98/79/EC. RUO reagents are intended for research use only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor, Pacific Blue and Pacific Orange are trademarks of Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corporation) Cy and CyDye are registered trademarks of GE Healthcare. The BD FACSCanto trademark is property of Becton, Dickinson and Company. 5

7 List of Reagents Immunophenotyping Single Color Reagents () Marker Fluorochrome Quantity Catalog No. Page CD3 PE 100 tests ED CD3 PerCP 100 tests ED CD4 FITC 100 tests ED CD4 PE 100 tests ED CD8 FITC 100 tests ED CD8 PE 100 tests ED CD14 FITC 100 tests ED CD14 PE 100 tests ED CD16 PE 100 tests ED CD19 PE 100 tests ED CD20 FITC 100 tests ED CD45 FITC 100 tests ED CD45 PE-Cy tests ED7067* 9 CD45 PerCP 100 tests ED CD56 PE 100 tests ED CD63 FITC 100 tests ED HLA-DR PE 100 tests ED Color Combinations () Markers Fluorochromes Quantity Catalog No. Page CD3 / CD4 FITC / PE 50 tests ED CD3 / CD8 FITC / PE 50 tests ED CD4 / CD8 FITC / PE 50 tests ED CD3 / CD19 FITC / PE 50 tests ED CD3 / CD16+CD56 FITC / PE 50 tests ED CD3 / HLA-DR FITC / PE 50 tests ED CD45 / CD14 FITC / PE 50 tests ED Negative Control FITC / PE 50 tests ED Color Combinations () Markers Fluorochromes Quantity Catalog No. Page CD4 / CD8 / CD3 FITC / PE / PE-Cy 5 50 tests ED7058* 18 CD4 / CD8 / CD3 FITC / PE / PerCP 50 tests ED7059* 19 CD3 / CD16+CD56 / CD45 FITC / PE / PerCP 50 tests ED CD3 / CD19 / CD45 FITC / PE / PerCP 50 tests ED CD3 / CD4 / CD45 FITC / PE / PerCP 50 tests ED CD3 / CD8 / CD45 FITC / PE / PerCP 50 tests ED *Available since May 1, 2014

8 4 Color Combinations () Markers Fluorochromes Quantity Catalog No. Page CD3 / CD8 / CD45 / CD4 FITC / PE / PerCP / APC 50 tests ED CD3 / CD16+CD56 / CD45 / CD19 FITC / PE / PerCP / APC 50 tests ED CD3 / CD8 / CD45 / CD4 PE-Cy 5 / PE / FITC / PE-Cy 7 50 tests ED7062* 26 CD3 / CD16+CD56 / CD45 / CD19 PE-Cy 5 / PE / FITC / PE-Cy 7 50 tests ED7063* 27 6 Color Combination () Markers Fluorochromes Quantity Catalog No. Page CD3 / CD16+CD56 / CD45 / FITC / PE / PerCP-Cy 5.5 / 50 tests ED7049* CD4 / CD19 / CD8 PE-Cy 7 / APC / APC- Cy 7 Lysing Solutions () Name Note Quantity Catalog No. Page EXCELLYSE I Two-step no-wash lysing 30 ml ( tests) ED protocol 100 ml ( tests) ED7065 EXCELLYSE Easy One-step no-wash lysing protocol 100 ml ( tests) ED Allergy Flow Cytometry Kit () Name Note Quantity Catalog No. Page BasoFlowEx Kit Basophil activation test (BAT) 100 tests ED Phagocytosis Flow Cytometry Kit () Name Note Quantity Catalog No. Page FagoFlowEx Kit Detection of oxidative burst defects 100 tests ED *Available since May 1,

9 Immunophenotyping Immunophenotyping is based on the specific binding of monoclonal antibodies to the antigenic determinants expressed on the surface of leukocytes. Blood samples are incubated with a mixture of fluorochrome labeled monoclonal antibodies, and red blood cell lysis is performed. Afterwards, unaffected leukocytes are subjected to analysis by a flow cytometer. The monoclonal antibodies are labeled with different fluorochromes which are excited by laser beams and their light emissions are collected and analyzed by the flow cytometer. The fluorescence intensity differences enable the separation of cell subsets based on the expression of analyzed antigens. Currently we offer pre-mixed combinations (2 6 colors) of fluorochrome-conjugated monoclonal antibodies (KOMBITEST reagents), as well as individual single color monoclonal antibody reagents. Spectral characteristics of fluorochromes contained in our IVD reagents for immunophenotyping: Excitation Emission max. FITC 488 nm 520 nm PE 488 nm 575 nm PE-Cy nm 664 nm PerCP 488 nm 675 nm PerCP-Cy nm 692 nm PE-Cy nm 774 nm APC 633 nm 660 nm APC-Cy nm 774 nm 8

10 Single color reagents Immunophenotyping Single Color Reagents 100 tests (2 ml) Specificity Fluorochrome Clone Isotype Catalog No. CD3 PE MEM-57 IgG2a ED7002 CD3 PerCP MEM-57 IgG2a ED7027 CD4 FITC MEM-241 IgG1 ED7013 CD4 PE MEM-241 IgG1 ED7003 CD8 FITC MEM-31 IgG2a ED7004 CD8 PE MEM-31 IgG2a ED7014 CD14 FITC MEM-15 IgG1 ED7028 CD14 PE MEM-15 IgG1 ED7015 CD16 PE LNK16 IgG1 ED7016 CD19 PE LT19 IgG1 ED7017 CD20 FITC LT20 IgG2a ED7005 CD45 FITC MEM-28 IgG1 ED7018 CD45 PE-Cy 5 MEM-28 IgG1 ED7067* CD45 PerCP MEM-28 IgG1 ED7029 CD56 PE MEM-188 IgG2a ED7019 CD63 FITC MEM-259 IgG1 ED7031 HLA-DR PE MEM-12 IgG1 ED7020 *Available since May 1,

11 Immunophenotyping 2-color reagents KOMBITEST CD3 FITC / CD4 PE ED7050 Specificity: CD3 CD4 Fluorochrome FITC PE Clone UCHT1 MEM-241 Isotype Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD3 FITC / CD4 PE is designed for percentage enumeration of mature human helper/inducer T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD4 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot CD4 PE vs. CD3 FITC. 10

12 KOMBITEST CD3 FITC / CD8 PE 2-color reagents Immunophenotyping ED7051 Specificity CD3 CD8 Fluorochrome FITC PE Clone UCHT1 MEM-31 Isotype Mouse IgG1 Mouse IgG2a λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD3 FITC / CD8 PE is designed for percentage enumeration of mature human suppressor/cytotoxic T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD8 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 FITC. 11

13 Immunophenotyping 2-color reagents KOMBITEST CD4 FITC / CD8 PE ED7052 Specificity: CD4 CD8 Fluorochrome FITC PE Clone MEM-241 MEM-31 Isotype Mouse IgG1 Mouse IgG2a λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD4 FITC / CD8 PE is designed for percentage enumeration of mature human helper/inducer (CD4 + ) and suppressor/cytotoxic (CD8 + ) lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD4 FITC / CD8 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot CD8 PE vs. CD4 FITC. 12

14 KOMBITEST CD3 FITC / CD19 PE 2-color reagents Immunophenotyping ED7053 Specificity CD3 CD19 Fluorochrome FITC PE Clone UCHT1 4G7 Isotype Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD3 FITC / CD19 PE is designed for percentage enumeration of mature human T and B lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD19 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot CD19 PE vs. CD3 FITC. 13

15 Immunophenotyping 2-color reagents KOMBITEST CD3 FITC / CD16+CD56 PE ED7054 Specificity: CD3 CD16 CD56 Fluorochrome FITC PE Clone UCHT1 3G8 MEM-188 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG2a λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD3 FITC / CD16+CD56 PE is designed for percentage enumeration of mature human natural killer (NK) lymphocytes and T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD16+CD56 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot CD16+CD56 PE vs. CD3 FITC. 14

16 KOMBITEST CD3 FITC / HLA-DR PE 2-color reagents Immunophenotyping ED7055 Specificity CD3 HLA-DR Fluorochrome FITC PE Clone UCHT1 MEM-12 Isotype Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD3 FITC / HLA-DR PE is designed for percentage enumeration of mature human activated T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / HLA-DR PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot HLA-DR PE vs. CD3 FITC. 15

17 Immunophenotyping 2-color reagents KOMBITEST CD45 FITC / CD14 PE ED7056 Specificity CD45 CD14 Fluorochrome FITC PE Clone MEM-28 MEM-15 Isotype Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST CD45 FITC / CD14 PE is designed for establishing the optimal lymphocyte gate for immunophenotyping of erythrocyte-lysed whole blood using flow cytometry. Monocytes Granulocytes Eosinophils Debris Lymphocytes Whole blood sample stained with KOMBITEST CD45 FITC / CD14 PE, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows leukocytes in a dot-plot CD14 PE vs. CD45 FITC. 16

18 2-color reagents Immunophenotyping KOMBITEST FITC / PE Negative Control ED7032 Specificity Undefined epitope on a plant pathogen Fluorochrome FITC PE Clone PPV-04 PPV-04 Isotype Mouse IgG2a Mouse IgG2a λ excitation 488 nm 488 nm Emission max. 525 nm 575 nm The reagent KOMBITEST FITC / PE Negative Control is designed for setting a boundary between negatively and positively stained lymphocytes when peripheral blood samples are analyzed using two-color (FITC / PE) KOMBITEST reagents. Whole blood sample stained with KOMBITEST FITC / PE Negative Control, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Figure shows lymphocytes in a dot-plot PE vs. FITC. 17

19 Immunophenotyping 3-color reagents KOMBITEST CD4 FITC / CD8 PE / CD3 PE-Cy 5 ED7058 Specificity: CD4 CD8 CD3 Fluorochrome FITC PE PE-Cy 5 Clone MEM-241 MEM-31 UCHT1 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 664 nm The reagent KOMBITEST CD4 FITC / CD8 PE / CD3 PE-Cy 5 is designed for percentage enumeration of mature human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T-lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD4 FITC / CD8 PE / CD3 PE-Cy 5, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Left figure shows lymphocytes in a dot-plot CD4 FITC vs. CD3 PE-Cy 5. Right figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 PE-Cy This product is available since May 1, 2014

20 3-color reagents Immunophenotyping KOMBITEST CD4 FITC / CD8 PE / CD3 PerCP ED7059 Specificity: CD4 CD8 CD3 Fluorochrome FITC PE PerCP Clone MEM-241 MEM-31 UCHT1 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 670 nm The reagent KOMBITEST CD4 FITC / CD8 PE / CD3 PerCP is designed for percentage enumeration of mature human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T-lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD4 FITC / CD8 PE / CD3 PerCP, lyzed using EXCELLYSE I and washed. Data acquired using BD FACSCanto. Left figure shows lymphocytes in a dot-plot CD4 FITC vs. CD3 PerCP. Right figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 PerCP. This product is available since May 1,

21 Immunophenotyping 3-color reagents KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP ED7060 Specificity: CD3 CD16 CD56 CD45 Fluorochrome FITC PE PerCP Clone UCHT1 3G8 MEM-188 MEM-28 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 664 nm The reagent KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP is designed for percentage enumeration of mature human natural killer (NK) lymphocytes and T-lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocyte gating. Right figure shows lymphocytes in a dot-plot CD16+CD56 PE vs. CD3 FITC. 20

22 KOMBITEST CD3 FITC / CD19 PE / CD45 PerCP 3-color reagents Immunophenotyping ED7061 Specificity: CD3 CD19 CD45 Fluorochrome FITC PE PerCP Clone UCHT1 4G7 MEM-28 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 670 nm The reagent KOMBITEST CD3 FITC / CD19 PE / CD45 PerCP is designed for percentage enumeration of mature human T and B lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD19 PE / CD45 PerCP and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocyte gating. Right figure shows lymphocytes in a dot-plot CD19 PE vs. CD3 FITC. 21

23 Immunophenotyping 3-color reagents KOMBITEST CD3 FITC / CD4 PE / CD45 PerCP ED7047 Specificity: CD3 CD4 CD45 Fluorochrome FITC PE PerCP Clone UCHT1 MEM-241 MEM-28 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 670 nm The reagent KOMBITEST CD3 FITC / CD4 PE / CD45 PerCP is designed for identification and enumeration of mature human T lymphocytes (CD3+) and mature human helper/inducer (CD3+ CD4+) T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. SSC-A Whole blood sample stained with KOMBITEST CD3 FITC / CD4 PE / CD45 PerCP and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocyte gating. Right figure shows lymphocytes in a dot-plot CD4 PE vs. CD3 FITC. 22

24 KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP 3-color reagents Immunophenotyping ED7048 Specificity: CD3 CD8 CD45 Fluorochrome FITC PE PerCP Clone UCHT1 MEM-31 MEM-28 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 λ excitation 488 nm 488 nm 488 nm Emission max. 525 nm 575 nm 670 nm The reagent KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP is designed for identification and enumeration of mature human T lymphocytes (CD3+) and suppressor/cytotoxic (CD3+ CD8+) T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocyte gating. Right figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 FITC. 23

25 Immunophenotyping 4-color reagents KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP /CD4 APC ED7045 Specificity: CD3 CD8 CD45 CD4 Fluorochrome FITC PE PerCP APC Clone UCHT1 MEM-31 MEM-28 MEM-241 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm 633 nm Emission max. 525 nm 575 nm 670 nm 660 nm The reagent KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP / CD4 APC is designed for identification and enumeration of mature human helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. The helper/suppressor ratio (CD4+ / CD8+) may also be determined. Whole blood sample stained with KOMBITEST CD3 FITC / CD8 PE / CD45 PerCP / CD4 APC and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 FITC. Right figure shows lymphocytes in a dot-plot CD4 APC vs. CD3 FITC. 24

26 4-color reagents Immunophenotyping KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP / CD19 APC ED7046 Specificity: CD3 CD16 CD56 CD45 CD19 Fluorochrome FITC PE PerCP APC Clone UCHT1 3G8 MEM-188 MEM-28 4G7 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm 633 nm Emission max. 525 nm 575 nm 670 nm 660 nm The reagent KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP / CD19 APC is designed for identification and enumeration of human T, natural killer, and B lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP / CD19 APC and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows lymphocytes in a dot-plot CD19 APC vs. CD3 FITC. Right figure shows lymphocytes in a dot-plot CD16+CD56 PE vs. CD3 FITC. 25

27 Immunophenotyping 4-color reagents KOMBITEST CD3 PE-Cy 5 / CD8 PE / CD45 FITC / CD4 PE-Cy 7 ED7062 Specificity: CD3 CD8 CD45 CD4 Fluorochrome PE-Cy 5 PE FITC PE-Cy 7 Clone UCHT1 MEM-31 MEM-28 MEM-241 Isotype Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm 488 nm Emission max. 664 nm 575 nm 525 nm 774 nm The reagent KOMBITEST CD3 PE-Cy 5 / CD8 PE / CD45 FITC / CD4 PE-Cy 7 is designed for identification and enumeration of mature human helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. The helper/suppressor ratio (CD4+ / CD8+) may also be determined. Whole blood sample stained with KOMBITEST CD3 PE-Cy 5 / CD8 PE / CD45 FITC / CD4 PE-Cy 7, lyzed using EXCELLYSE Easy and washed. Data acquired using Beckman Coulter Cytomics FC 500. Left figure shows lymphocytes in a dot-plot CD8 PE vs. CD3 PE-Cy 5. Right figure shows lymphocytes in a dot-plot CD4 PE-Cy 7 vs. CD3 PE-Cy This product is available since May 1, 2014

28 4-color reagents Immunophenotyping KOMBITEST CD3 PE-Cy 5 / CD16+CD56 PE / CD45 FITC / CD19 PE-Cy 7 ED7063 Specificity: CD3 CD16 CD56 CD45 CD19 Fluorochrome PE-Cy 5 PE FITC PE-Cy 7 Clone UCHT1 3G8 MEM-188 MEM-28 4G7 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 λ excitation 488 nm 488 nm 488 nm 488 nm Emission max. 664 nm 575 nm 525 nm 774 nm The reagent KOMBITEST CD3 PE-Cy 5 / CD16+CD56 PE / CD45 FITC / CD19 PE-Cy 7 is designed for identification and enumeration of human T, natural killer, and B lymphocytes in erythrocyte-lysed whole blood using flow cytometry. Whole blood sample stained with KOMBITEST CD3 PE-Cy 5 / CD16+CD56 PE / CD45 FITC / CD19 PE-Cy 7, lyzed using EXCELLYSE Easy and washed. Data acquired using Beckman Coulter Cytomics FC 500. Left figure shows lymphocytes in a dot-plot CD16+CD56 PE vs. CD3 PE-Cy 5. Right figure shows lymphocytes in a dot-plot CD19 PE-Cy 7 vs. CD3 PE-Cy 5. This product is available since May 1,

29 6-color reagents Immunophenotyping KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP-Cy 5.5 / CD4 PE-Cy 7 / CD19 APC / CD8 APC-Cy 7 ED7049 Specificity CD3 CD16 CD56 CD45 CD4 CD19 CD8 Fluorochr. FITC PE PerCP-Cy 5.5 PE-Cy 7 APC APC-Cy 7 Clone UCHT1 3G8 MEM-188 MEM-28 MEM-241 4G7 MEM-31 Isotype Mouse IgG1 Mouse IgG1 Mouse IgG2a Mouse IgG1 Mouse IgG1 Mouse IgG1 Mouse IgG2a λ excitation 488 nm 488 nm 488 nm 488 nm 633 nm 633 nm Emission m. 525 nm 575 nm 692 nm 774 nm 660 nm 774 nm The reagent KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP-Cy 5.5 / CD4 PE-Cy 7 / CD19 APC / CD8 APC-Cy 7 is designed for identification and enumeration of mature human B lymphocytes, natural killer cells, T lymphocytes, helper/inducer (CD3+CD4+) T lymphocytes, and suppressor/cytotoxic (CD3+CD8+) T lymphocytes in erythrocyte-lysed whole blood using flow cytometry. 28 This product is available since May 1, 2014

30 Lysing solutions Immunophenotyping fig.1 CD3- lymphocytes fig. 2a fig. 2 CD3+ lymphocytes fig. 2b Whole blood sample stained with KOMBITEST CD3 FITC / CD16+CD56 PE / CD45 PerCP- Cy 5.5 / CD4 PE- Cy 7 / CD19 APC / CD8 APC- Cy 7 and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Figure 1 shows lymphocytes gating. Figure 2 shows CD3+ and CD3- lymphocyte gating. Figure 2a shows CD3- lymphocytes in a dot-plot CD19 APC vs. CD16+CD56 PE. Figure 2b shows CD3+ lymphocytes in a dot-plot CD4 PE-Cy 7 vs. CD8 APC-Cy 7. 29

31 Immunophenotyping Lysing solutions EXCELLYSE I ED ml ( tests) ED ml ( tests) The EXCELLYSE I lysing solution permits red blood cell lysis following antibody staining of human peripheral blood leukocytes using both lyse/wash and lyse/no wash protocol. This solution is a ready to use reagent provided as 30 ml or 100 ml package. Features for lyse/no-wash protocol: 2-step lysing procedure Excellent separation of lymphocytes and debris in SSC vs. FSC dot plot Granulocytes Granulocytes Debris Debris Monocytes Monocytes Lymphocytes Lymphocytes Whole blood sample stained with CD45 PerCP antibody and lyzed using EXCELLYSE I (no wash protocol). Data acquired using BD FACSCanto. Left figure shows leukocytes in a dot-plot SSC-A vs. FSC-H. Right figure shows leukocytes in a dot-plot SSC-A vs. CD45 PerCP. 30

32 Lysing solutions Immunophenotyping EXCELLYSE Easy ED ml ( tests) The EXCELLYSE Easy lysing solution permits red blood cell lysis following antibody staining of human peripheral blood leukocytes using both lyse/wash and lyse/no wash protocol. This solution is provided as 100 ml of 10 concentrated reagent and must be diluted 1:10 with deionized water prior to use. Features for lyse/no wash protocol: 1-step lysing procedure Suitable for lymphocyte gating via CD45 staining Granulocytes Granulocytes Debris Monocytes Debris Monocytes Lymphocytes Lymphocytes Whole blood sample stained with CD45 PerCP antibody and lyzed using EXCELLYSE Easy (no wash protocol). Data acquired using BD FACSCanto. Left figure shows leukocytes in a dot-plot SSC-A vs. FSC-H. Right figure shows leukocytes in a dot-plot SSC-A vs. CD45 PerCP. 31

33 Allergy Allergic hypersensitivity a challenge to modern diagnostics A rapid increase in cases of allergic hypersensitivity in developed countries has been observed over the last decades, which indicates that this condition is about to become a serious social problem. The physiological regulation of allergic mechanisms depends on maintaining a delicate balance between the processes responsible for optimal modulation of immune strategies in an organism. This balance can be impaired by many factors, affecting various levels of immune regulation. To be able to diagnose what the exact cause of a particular patient s condition is, and what the optimal therapy would be, is still only achievable in rare instances. Advantages of the Basophil Activation Test (BAT) in diagnosing allergies When diagnosing allergies, the crucial task is determining the causal allergen(s). The commonly used method of skin prick tests (SPT) is not optimal due to its low reliability and the risk of further aggravating the condition. More suitable are in vitro approaches, such as the determination of specific IgE in the serum (sige) or the analysis of basophil activation markers. In most cases, the specificity of sige detection is high enough, but the sensitivity is usually only about 75%, and in cases of some allergens (mainly food and drug) both sensitivity and specificity of sige detection are substantially lower. Basophil Activation Test (BAT), however, is known not only for very high specificity, but also for high sensitivity, even in cases of many problematic antigens. This test is based on the detection of basophil activation markers using flow cytometry. The BasoFlowEx Kit is based on the principles of the BAT. Its use is simple and suitable for every laboratory with basic equipment and with a flow cytometer, which uses blue laser excitation (488 nm) and detection channels for FITC and PE. 32

34 Allergy ED7043 Reagents provided: ED Stimulation Buffer ED Stimulation Control ED Staining Reagent ED Lysing Solution 100 tests 5 lyophilized vials 1 vial is intended for stimulation of 20 tubes 2 lyophilized vials 1 vial is intended for stimulation of 25 positive controls 1 vial containing 2 ml of premixed antibody cocktail: anti-cd63, FITC labeled + anti-cd203c, PE labeled 30 ml The BasoFlowEx Kit is intended for flow cytometry examination of IgE-mediated allergic reactions via the analysis of CD63 antigen surface exposure on basophils in human heparinized whole blood upon allergen stimulation. Commercially available allergens (e.g. used for skin prick tests) can be used for stimulation. Left: Delimitation of basophil population (CD203c pos /SSC low ). Right: Histogram of allergen-stimulated basophils. Left: Histogram of negative control basophils. Right: Histogram of positive control basophils. 33

35 Phagocytosis Phagocytosis mechanism Phagocytosis, the process where specialised cells of the immune system kill and decompose microorganisms (extracellular bacteria), is fundamental to innate non -specific human immunity. It is preceded by chemotactic migration of the phagocytes (mainly neutrophilic granulocytes) into the site of inflammation. Then the foreign particles are recognized, ingested (forming phagosome and phagolysosome), and finally destructed by oxygen -independent and oxygen- dependent mechanisms. The latter ones (the oxidative burst ) involve a release of reactive oxygen radicals, hydrogen peroxide, hydroxyl radical, and hypochlorite. In this process the key enzymes are the NADPH oxidase and myeloperoxidase, whose malfunction leads to impaired bactericidal mechanisms. Why to survey phagocytic activity? To confirm or exclude defective phagocytosis, primary and secondary immunodeficiences. When diagnosing primary immunodeficiences, the examination of phagocytic activity is the first method of choice. Clinical symptoms of hereditary immunodeficiences usually manifest in early childhood, however, the less severe forms can sometimes be manifested later in adulthood. Without early and adequate therapy, these illnesses are life threatening. 34

36 Phagocytosis Reagents provided: ED tests ED E.coli 5 vials containing lyophilized E.coli bacteria 1 vial is intended for stimulation of 20 tubes ED DHR123 5 vials containing lyophilized Dihydrorhodamine vial is intended for staining of 60 tubes ED Stimulation Control 5 vials containing lyophilized PMA (Phorbol 12-myristate 13-acetate) 1 vial is intended for stimulation of 20 positive controls ED Lysing Solution 15 ml The FagoFlowEx Kit is intended for the examination of phagocytic activity of neutrophil granulocytes by measuring the respiratory (oxidative) burst after their stimulation with E.coli bacteria in human heparinized whole blood using flow cytometry. red: stimulated sample blue: negative control Left: Patient with myeloperoxidase deficiency (SI = 8.2). Right: Donor without defect of respiratory burst (SI = 98). Male patient (left) with chronic granulomatous disease and his mother (right) carrying the X linked mutation of the NADPH oxidase. Male patient had SI = 13.6, mother had two granulocyte subpopulations differing in respiratory burst intensity: SI low = 13.9, SI high = 125. Stimulation index (SI) means the MFI ratio of positive granulocytes of E.coli stimulated sample and negative granulocytes of negative control sample. The stimulation index is used for approximate comparison of respiratory burst intensity between blood samples. 35

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