anti-Myeloperoxidase-C2 (FITC) and anti-CD22 (PE) antibody pair

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Antigen
Reactivity
Human
Host
Mouse
Clonality (Clone)
Monoclonal ()
Conjugate
FITC,PE
Application
Flow Cytometry (FACS)
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Purpose This product is optimised for use with FIX&PERM®.
Brand FIX&PERM®
Clone 8E6-RFB4
Isotype IgG1
Specificity Antibody MPO-C2 (clone 8E2) reacts with human myeloperoxidase (MPO) expressed by normal and malignant myelomonocytic cells. The CD22 mAb (clone RFB4) recognizes cytoplasmatic CD22 in precursor B-cells and surface as well cytoplasmatic CD22 on mature B-cells. In this COMBI-IC Reagent antibody 8E6 is conjugated to FITC, antibody RFB4 is conjugated to Phycoeythrin (PE).The sensitivity of MPO-C2/CD22 mAb is determined by staining well-defined blood samples from representative donors with serial-fold mAb dilutions to obtain a titration curve that allows relating the mAb concentration to the percentage of stained cells and geometric MFI (mean fluorescence intensity). For this purpose, a mAb-concentration range is selected to include both the saturation point (i.e. the mAb dilution expected to bind all epitopes on the target cell) and the detection threshold (i.e. the mAb dilution expected to represent the least amount of mAb needed to detect an identical percentage of cells). In practice, 50 µL of leukocytes containing 10^7 cells/mL are stained with 20µL mAb of various dilutions to obtain a titration curve and to identify the saturation point and detection threshold. The final concentration of the product is then adjusted to be at least 3-fold above the detection threshold. In addition and to control lot-to-lot variation, the given lot is compared and adjusted to fluorescence standards with defined intensity.
Characteristics Mildly fixes cells, preserving their flow cytometric scatter characteristics
Allows simultaneous characterisation of both intracellular and cell surface markers
Rapid technique - whole procedure can be carried out in less than one hour, ready
for immediate analysis or storage for 24 hours
Stringent QC procedures - the quality of each lot is determined using well-defined
blood samples and subsequent comparison of scatter characteristics of obtained
leukocyte populations, ensuring consistent and reliable results lot after lot
A range of intracellular antibodies with optimised protocols for use with FIX&PERM®
FIX&PERM® is a simple procedure making use of two reagents. Reagent A gently fixes cells, while Reagent B permeabilizes them. The specific formulations reduce background and allow simultaneous addition of permeabilization medium and fluorochrome labelled antibodies, allowing staining of intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins, etc.
Purification Purified by Affinity Chromatography
Components COMBI IC Reagent: Mouse anti Myeloperoxidase-C2 (FITC) and Mouse anti CD22 (PE)
Sub Type Cocktail
Molecular Weight 14 kDa
Application Notes Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX&PERM- (ABIN1741575) intracellular MPO-C2 and CD22 can be easily stained in cell suspensions. - For each sample to be analyzed add 50 µL of whole blood, bone marrow or mononuclear cell suspension in a 5 mL tube - Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature) - Incubate for 15 minutes at room temperature - Add 5 mL phosphate buffered saline and centrifuge cells for 5 minutes at 300 g - Remove supernatant and add to cell pellet 100 µL Reagent B (Permeabilization Medium) and 20 µL of the MPO-C2/CD22 COMBI-IC monoclonal antibody conjugate - Vortex at low speed for 1-2 seconds - Incubate for 15 minutes at room temperature - Wash cells with phosphate buffered saline as described above - Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 mL 1.0 % formaldehyde and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours.
Comment

Myeloperoxidase (MPO) is a glycoprotein present in the azurophil (primary) granules of myeloid cells, which appears in the myeloblast stage of myeloid cell differentiation. MPO is he most common functional protein of myeloid cells and is involved in the inflammatory response. It helps to kill microbes by breaking down peroxide in the presence of halide ions, contributing to the bactericidal function of granulocytes. The primary translation product of MPO undergoes glycosylation with production of the 89 kDa heme-free apopro-MPO form followed by incorporation of heme and conversion into the enzymatically active pro-MPO form. Subsequently, pro-MPO becomes targeted to azurophil granules where final processing occurs to produce mature dimeric MPO consisting of the 59-64 kDa MPO α-chain and the 14 kDa MPO β-chain. Precursor B-cells are surface-CD22 negative, but cytoplasmic CD22 positive. Mature B-lymphocytes.express CD22 also on their surface. The combined staining for MPO and CD22 allows the distinction of mature/immature myeloid cells and B-lymphocytes. The MPO-C2/CD22 COMBI-IC mAb permits the identification and enumeration of normal and malignant myelomonocytic cells and B lymphoid commited cells in human blood and bone marrow using flow cytometry. Results must be put within the context of other diagnostic tests as well as the clinical history of the patient by a certified professional before final interpretation. Analyses performed with this antibody should be paralleled by positive and negative controls.

Assay Procedure

Procedure: For each sample to be analysed use 50 µL of whole blood, bone marrow or mononuclear cell suspension in a 5 mL tube
Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature)
Incubate for 15 minutes at room temperature
Add 5 mL phosphate buffered saline and centrifuge cells for 5 minutes at 300 g
Remove supernatant and add to cell pellet 100 µL Reagent B (Permeabilization
Medium) and 20 µL of the appropriate monoclonal antibody conjugate
Vortex at low speed for 1-2 seconds
Incubate for 15 minutes at room temperature
Wash cells with phosphate buffered saline as described above
Remove supernatant and re-suspend cells in sheath fluid for immediate analysis or re-suspend cells in 0.5 mL 1.0 % formaldehyde and store at 2-8°C in the dark
Analyse fixed cells within 24 hours

Restrictions For Research Use only
Buffer PBS pH 7.2, 1 mg/mL BSA, 0.1 % sodium azide
Preservative Sodium azide
Precaution of Use This reagent contains sodium azide. To avoid the development of hazardous conditions, reagents containing azide should be diluted in running water prior to be discarded. Similar to the work with other biological products, proper handling procedures are recommended.
Handling Advice Do not freeze and protect from prolonged exposure to light.
Storage 4 °C
Storage Comment These reagents should be stored at 2-8°C Stability of the reagent: Please refer to the expiry date printed onto the vial. The use of the reagent after the expiration date is not recommended.
Product cited in: Braylan, Orfao, Borowitz, Davis: "Optimal number of reagents required to evaluate hematolymphoid neoplasias: results of an international consensus meeting." in: Cytometry, Vol. 46, Issue 1, pp. 23-7, 2001 (PubMed).

Koníková, Glasová, Kusenda, Babusíková: "Intracellular markers in acute myeloid leukemia diagnosis." in: Neoplasma, Vol. 45, Issue 5, pp. 282-91, 1999 (PubMed).

Andersson, Hellman, Gullberg, Olsson: "The role of the propeptide for processing and sorting of human myeloperoxidase." in: The Journal of biological chemistry, Vol. 273, Issue 8, pp. 4747-53, 1998 (PubMed).

Lanza, Latorraca, Moretti, Castagnari, Ferrari, Castoldi: "Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells." in: Cytometry, Vol. 30, Issue 3, pp. 134-44, 1997 (PubMed).

Madrigal, Belich, Benjamin, Little, Hildebrand, Mann, Parham: "Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells." in: The Journal of experimental medicine, Vol. 174, Issue 5, pp. 1085-95, 1991 (PubMed).

Catovsky, Matutes, Buccheri, Shetty, Hanslip, Yoshida, Morilla: "A classification of acute leukaemia for the 1990s." in: Annals of hematology, Vol. 62, Issue 1, pp. 16-21, 1991 (PubMed).

Janossy, Coustan-Smith, Campana: "The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases." in: Leukemia, Vol. 3, Issue 3, pp. 170-81, 1989 (PubMed).

Boué, Lebien: "Structural characterization of the human B lymphocyte-restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein-Barr virus receptor)." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 140, Issue 1, pp. 192-9, 1988 (PubMed).

Murao, Stevens, Ito, Huberman: "Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, Issue 4, pp. 1232-6, 1988 (PubMed).

Mason, Stein, Gerdes, Pulford, Ralfkiaer, Falini, Erber, Micklem, Gatter: "Value of monoclonal anti-CD22 (p135) antibodies for the detection of normal and neoplastic B lymphoid cells." in: Blood, Vol. 69, Issue 3, pp. 836-40, 1987 (PubMed).

Koeffler, Ranyard, Pertcheck: "Myeloperoxidase: its structure and expression during myeloid differentiation." in: Blood, Vol. 65, Issue 2, pp. 484-91, 1985 (PubMed).