COMBI IC Reagent: Mouse anti Myeloperoxidase-C2 (FITC) and Mouse anti CD22 (PE)


Catalogue number: GIC-214

Clone8E6 and RFB4
IsotypeIgG1
Product Type Primary Antibodies
Units1 ml
HostMouse
Species reactivity Human
Application Flow cytometry
Background
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. If unexpected results are obtained which cannot be attributed to differences in laboratory procedures, please contact us.

Product
1ml of FITC-conjugated anti Myeloperoxidase-C2 (clone 8E6) and PE-conjugated anti CD22 (cloneRFB4) in PBS pH 7.2, 1% BSA, and 0.05% NaN3, approximately 50 tests.

Applications
Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX&PERM (Cat. No. GAS-002) 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 5ml tube - Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature) - Incubate for 15 minutes at room temperature - Add 5ml 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.

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.

Storage
Nordic-MUbio monoclonal antibody reagents contain optimal concentrations of affinity-purified antibody. For stability reasons this monoclonal antibody solution contains sodium azide. These reagents should be stored at 2-8°C (DO NOT FREEZE!) and protected from prolonged exposure to light. 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.

References
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11. Li, J. L., Shen, G. L., Ghetie, M. A., May, R. D., Till, M., Ghetie, V., Uhr, J. W., Janossy, G., Thorpe, P. E., Amlot, P. & et al. (1989) Cell Immunol 118, 85-99.
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13. Murao, S., Stevens, F. J., Ito, A. & Huberman, E. (1988) Proc Natl Acad Sci U S A 85, 1232-6.

Caution
For professional users only. 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.