Cd79a Antibodies,Package Insert

CD79a Antibodies For in vitro diagnostic use

s Use of the reagents is restricted to trained and qualified personnel only. s All biological specimens and all materials that come into contact with Miltenyi Biotec B.V. & Co. KG blood and blood products must be treated as infectious material. Friedrich-Ebert-Str. 68 Regulations for the treatment and disposal of infectious material must 51429 Bergisch Gladbach be followed. Germany +49 2204 8306-8484 s Reagents contain sodium azide (NaN3), a chemical highly toxic in www.miltenyibiotec.com pure form. However, at product concentrations, it is not classified as hazardous. Sodium azide may react with lead and copper plumbing to form highly explosive buildups of metal azides. Upon disposal, 1. General information flush with large volumes of water to prevent metal azide build-up in plumbing. Safety guidelines must be observed. Intended use HM47 reacts with human CD79a. The fluorescently labeled CD79a antigen can s For material required but not provided the manufacturers be detected by flow cytometry. recommendations and safety regulations must be followed. Reagents and contents Directions of the package insert must be followed to obtain accurate Monoclonal CD79a antibody conjugates and reproducible results.

Product Volume REF 4. Application 100 Reagents can be used for immunophenotyping by flow cytometry. Abnormal CD79a-PE for 100 tests 1 mL 170-078-035 numbers of cells expressing this antigen or aberrant expression levels of the antigen can be expected in some disease states. It is important to understand 2. Technical data and background information the normal expression pattern for this antigen and its relationship to expression of other relevant antigens in order to perform appropriate analysis. Antigen CD79a Expression of CD79a may be used as aid to diagnostic in the characterization Clone HM47 of samples from individuals suspected with hematologic neoplasia. Isotype mouse IgG1κ 5. Materials required but not provided Alternative names Igα, MB1 - Disposable capped polystyrene tubes, 12×75 mm of antigen - Buffer: Prepare a solution containing phosphate-buffered saline (PBS), Product formulation Antibodies are supplied in buffer containing pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting stabilizer and 0.05% sodium azide. e.g. MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091‑222). Keep buffer cold (+2 °C to +8 °C). +8˚C - Fixation and permeabilization solutions for the fixation and +2˚C Store at +2 °C to +8 °C. Do not freeze. permeabilization of cells: e.g. Inside Stain Kit e.g. (# 130-090-477), or equivalent Store protected from light. - Double-distilled water - Micropipettes with disposable tips: variable micropipettes with volume ranges of 10–100 μL and 100–1000 μL The use-by date is indicated on the vial label. - Low speed centrifuge: minimum speed 300×g, with 12×75 mm tube carriers For in-use stability at +2 °C to +8 °C storage - Vortex mixer temperature refer to the use-by date indicated - Flow cytometer with appropriate laser and filter settings on the vial label. Do not use the reagent after the use-by date. 6. Protocol Expression pattern Principle of method: HM47 recognizes the cytoplasmic domain of human CD79a, which is also The antibody reagent provided enables the identification of a specific target known as mb-1 and Ig α. Along with CD79b (Ig β), CD79a is associated with cell type by flow cytometry. This technique is based on fluorochrome- surface Ig (sIg) to form the B cell antigen receptor complex. CD79a is a 47 conjugated antibodies binding to specific antigens expressed by the target kDa glycoprotein and comprises a single extracellular immunoglobulin cells. Incubating a sample of interest, e.g., peripheral blood mononuclear cells domain, a transmembrane domain, and a signaling intracellular domain (PBMC), with the provided antibody reagent leads to fluorescent staining of with immunoreceptor tyrosine-based activation motif (ITAM). CD79a/CD79b the cell type expressing the specific target antigen. Analysis of the sample heterodimer facilitates differentiation of pre-B cells from pro-B cells, surface is performed in a flow cytometer at a single-cell level. The analysis is based expression of sIg, signal transduction following antigen recognition and on the detection of characteristic light emission patterns emitted by the endocytosis of recognized antigens. Surface expression of CD79a diminishes fluorescently labeled antibody upon excitation with laser light. The collected in plasma cells, where it is mainly found as an intracellular molecule. CD79 data can be processed and analyzed using flow cytometry software. is considered a B cell–specific marker and is often used to identify the B cell lineage of acute lymphoblastic leukemia. Recent reports however suggest Important notes: expression of CD79a in T cell acute lymphoblastic leukemia. Individuals Under some conditions red blood cells may not lyse within 10 minutes. In this lacking CD79a have low/absent circulating pre-B cells and serum antibodies case extend lysis time to 20 minutes before centrifugation of samples. due to a block in B cell development. Exposure of reagents to temperatures below +2 °C and above +8 °C and to 3. Warnings and precautions light should be minimized during handling. s Analysis results obtained by use of the reagents shall never be the sole Sample requirements basis for classification of disease states. - Reagents can be used for determination of antigen-positive cells in whole blood samples by flow cytometry. Each cell source can have different s Interpretation of results is under the full responsibility of the user. storage conditions and limitations that should be considered prior to s For all handling, consideration of good laboratory practice (GLP) collection and analysis. For collection of patient samples European and regulations is recommended. national legislation must be followed. P/N: 33437/02 | Issued: 2020–05

page 1/2 - Whole blood samples should be stained within 24 hours. 9. Limitations Single color immunofluorescence provides only limited information and - Viability of the cells should be assessed and use of samples with at least is not the method of choice for comprehensive analysis of hematological 80% viable cells is suggested in order to minimize risk of erroneous results. malignancies. Multicolor immunophenotyping allows more precise definition - Cell count of white blood cells (WBC) should not exceed 5×107 cells/mL. of atypical cell populations. Therefore, multicolor analysis using relevant combinations of reagents is highly recommended. Note: If necessary dilute cell sample with PEB buffer. As reagents can be used in different combinations, each individual laboratory Protocol for intracellular staining needs to become familiar with the reactivity of each antibody in conjunction 1. Add 100 µL of whole blood to a 12x75 mm tube. with other markers in normal and abnormal samples. 2. Add 100 µL Inside Fix and incubate for 15 minutes at room temperature Use of monoclonal antibodies in patient treatment can interfere with (+20 − +25 °C). recognition of target antigens by this reagent. This should be considered when analyzing samples from patients treated in this fashion. Miltenyi Biotec 3. Add 1 mL of Inside Perm and incubate for 10 minutes at room has not characterized the effect of the presence of therapeutic antibodies on temperature. Centrifuge at 300x g for 5 minutes. Remove supernatant. the performance of this reagent. Resuspend cell pellet in 100 µL of Inside Perm and proceed to staining. Reagent data performance was collected typically with EDTA-treated blood. 4. Add 10 μL of fluorochrome-conjugated antibody to 100 µL of cell sample Reagent performance can be affected by the use of other anticoagulants. in a 12×75 mm tube. 5. Mix well and incubate for 15 minutes in the dark at room temperature 10. References (+20 °C to +25 °C). 1. van Dongen JJM et al: EuroFlow antibody panels for standardized m-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia Note: Higher temperatures and/or longer incubation times may lead to 2012; 26: 1908-197 non-specific cell labeling. Working on ice requires increased incubation 2. Rothe G, Schmitz G. Consensus protocol for the flow cytometric Immunophenotyping times. of hematopoietic malignancies. Leukemia1996; 10:877-895 6. Wash cells by adding 1−2 mL of buffer, centrifuge at 300×g for 3. Stelzer GT et al: U.S.-Canadian Consensus Recommendations on the Immunophenotypic 10 minutes. Remove supernatant. Analysis of Hematologic Neoplasia by Flow Cytometry: Standardization and Validation of Laboratory Procedures. Cytometry 1997; 30: 214–230 7. Resuspend cell pellet in a suitable amount of buffer and proceed to flow 4. Clinical and Laboratory Standards Institute (CLSI). Enumeration of Immunologically cytometric analysis. Store samples at +2 °C to +8 °C until analysis. Defined Cell Populations by Flow Cytometry; Approved Guideline - Second Edition. CLSI Note: Minimize exposure of samples to light. document H42-A2 (ISBN 1-56238-640-9) 2007 5. Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Quality control Neoplastic Hematolympoid Cells; Approved Guideline - Second Edition. CLSI document It is recommended to run regularly a control sample from a normal adult H43-A2 (ISBN 1-56238-635-2) 2007 specimen or commercially available whole blood control as a quality control 6. Lin, J. et al. (1992) The MB-1/B29 heterodimer couples the B cell antigen receptor to of the system. For excitation and emission data of fluorochrome conjugated multiple src family protein tyrosine kinases. J. Immunol. 149: 1548–1555. reagent please refer to chapter 8. 7. Mason, D. Y. et al. (1991) The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells. J. Immunol. 147(11): 2474–2482. 7. Performance characteristics 8. Minegishi, Y. et al. (1999) Mutations in Igalpha (CD79a) result in a complete block in B-cell development. J. Clin. Invest. 104(8): 1115–1121. Precision 9. Koyama, M. et al. (1997) CD79α/CD79β heterodimers are expressed on pro-B cell CD79a antibodies were tested by flow cytometry after fixation and surfaces without associated μ heavy chain. Int. Immunol. 9: 1767–1772. permeabilization of whole blood from healthy donors. Reproducibility was 10. Lai, R. et al. (2000) Flow cytometric detection of CD79a expression in T-Cell acute assessed by measuring the frequency of CD79a positive cells in replicate lymphoblastic leukemias. Am. J. Clin. Pathol. 113(6): 823–830. measurements performed by different operators using the same set of different donor samples. Precision was inferred from calculating the mean, 11. Glossary of symbols standard deviation and coefficient of variation of the frequency of positive cells. All values were within the acceptance criterion. Analytical specificity Manufacturer In-use stability Analytical specificity was evaluated by comparing clone HM47 to a relevant +8˚C reference clone of the same specificity. Reactivity towards the same antigen was inferred from the antibody blocking capacity or the staining Order number +2˚C Temperature limit diagonal observed during co-incubation of HM47 with the reference clone. Measurements were performed using different donor samples. All measurements were within the acceptance criterion. Part number Protect from sunlight

8. Excitation and emission data of fluorochrome conjugates European conformity Batch code approval Fluorochrome Excitation laser Excitation Emission (nm) maximum (nm) maximum (nm) In vitro diagnostic medical device Phone VioBlue® 405 400 452 VioGreen™ 405 388 520 Use-by date Website VioBright™ FITC 488 496 522 Consult instruction for FITC 488 495 520 use PE 488 or 561 565 578 PE-Vio®615 488 or 561 565 619 MACS, the Miltenyi Biotec logo, Vio, VioBlue, VioGreen and VioBright are registered PerCP 488 482 675 trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various PerCP-Vio®700 488 482 676 countries worldwide.

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