Product: CFTMBlue Anti-human CD52
Alternative names: CAMPATH-1.
Cat. Ref: 52CFB-100T
Reagent provided: 100 test (5μl/test)
Description: Monoclonal Mouse Anti-Human CD52 CFTMBlue is recommended for use in flow cytometry
for identification of thymocytes, lymphocytes, monocytes and macrophages. The conjugate is provided
in liquid form in buffer containing an antibody stabilizer solution and 0,09% NaN3, pH 7.2.
Clone: HI186
Isotype: IgG2b
Fluorochrome: CF405M (Abs/Em Max: 408/450 nm) US patent application no. 12/607,915
Direct replacement for: Pacific Blue dye®, BD HorizonTM V450)
Reactivity: React with CD52, also known as CAMPATH-1. It is expressed on thymocytes, lymphocytes,
monocytes and macrophages, but not on plasma cells, platelets or erythrocytes. This molecule is
expressed on lymphoid malignancies at variable levels and on epithelial cells of the epididymis and
seminal vesicles. Its functional role is not well known but reports indicate that CD52 is a good target for
complement-mediated cell lysis and antibody-mediated cellular cytotoxicity. Antibodies to CD52 have
been studied in lymphocyte depletion experiments.
Specificity: HI186 reacts with the human CD52 antigen, also known as CAMPATH-1. The CD52 antigen is
a remarkably small peptide that is heavily glycosylated, and attached to the cell surface membrane via a
GPI link. The apparent molecular mass of the antigen on SDS-PAGE is 25-29 kD. CD52 is expressed at
high density by lymphocytes, monocytes, eosinophils, thymocytes and macrophages. It is expressed by
most lymphoid derived malignancies, although expression on myeloma cells is variable.
Storage: Store in the dark at 2-8 °C. Do not use after expiration date stamped on vial. If unexpected
staining is observed which cannot be explained by variations in laboratory procedures and a problem
with the product is suspected, contact our Technical Services. (tech@immunostep.com).
Application: It is recommended for use in flow cytometry. This reagent is effective for direct
6
immunofluorescence staining of human tissue for flow cytometric analysis using 5 μl/10 cells.
Precautions:
1. The device is not intended for clinical use including diagnosis, prognosis, and monitoring of a disease
state, and it must not be used in conjunction with patient records or treatment.
2. This product contains sodium azide (NaN3), a chemical highly toxic in pure form. At product
concentrations, though not classified as hazardous, sodium azide may react with lead and copper
plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of
water to prevent metal azide build-up in plumbing.
Preparation:
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Transfer 100 μL of anticoagulated (EDTA) blood to a 12 x 75 mm polystyrene test tube (106 cells).
Add 5 μL of CD52 CF Blue and mix gently with a vortex mixer. The 5 μL is a guideline only; the
optimal volume should be determined by the individual laboratory.
The recommended negative control is a non-reactive CF Blue-conjugated antibody of the same
isotype.
Incubate in the dark at 4 °C for 30 minutes or at room temperature (20-25 °C) for 15 minutes.
Add 1,5 mL of Lysing Solution to each sample and mix gently with a vortex mixer. Incubate for
10 minutes at room temperature in the dark.
Centrifuge at 1000 x g for 5 minutes. Gently aspirate the supernatant and discard it leaving
approximately 50 μL of fluid.
Add 2 mL 0.01 mol/L PBS (It betters that it containing 2% bovine serum albumin) and
resuspend the cells by using a vortex mixer.
Centrifuge at 1000 x g for 5 minutes. Gently aspirate the supernatant and discard it leaving
approximately 50 μL of fluid.
Resuspend pellet in an appropriate fluid for flow cytometry, e.g. 0.3 mL PBS. The PBS should
contain 1% paraformaldehyde (fixative) if samples are not analysed the same day.
Revision Nº 2
Emission date: 2013-02-13
HT-0052-CFB-1
10. Analyse on a flow cytometer or store at 2-8 °C in the dark until analysis. Samples can be run up
to 24 hours after lysis.
Normal Peripheral Blood on a human donor
Staining of normal
human peripheral
blood cells with AntiHuman CD52 CFBlue
(Cat. 52CFB-100T).
Cells in the
Leukocytes gate were
used for analysis.
Cells were analyzed on a BD FACSAria™ II Flow Cytometer (Becton Dickinson, San Jose, CA), using Cell
Quest acquisition software.
FOR MORE INFORMATION, PLEASE VISIT OUR WEBSITE: www.immunostep.com
References:
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4.
Kanda Y, Oshima K, Asano-Mori Y, Kandabashi K, Nakagawa M, Sakata-Yanagimoto M, Izutsu K,
Hangaishi A, Tsujino S, Ogawa S, Motokura T, Chiba S, Hirai H. In vivo alemtuzumab enables
haploidentical human leukocyte antigen-mismatched hematopoietic stem-cell transplantation
without ex vivo graft manipulation. Transplantation. 2005 May 27;79(10):1351-7.
Lin TS, Flinn IW, Lucas MS, Porcu P, Sickler J, Moran ME, Lucas DM, Heerema NA, Grever MR, Byrd
JC. Filgrastim and alemtuzumab (Campath-1H) for refractory chronic lymphocytic leukemia.
Leukemia. 2005 Apr 28;
Popat U, Carrum G, May R, Lamba R, Krance RA, Heslop HE, Brenner MK. CD52 and CD45
monoclonal antibodies for reduced intensity hemopoietic stem cell transplantation from HLA
matched and one antigen mismatched unrelated donors. Bone Marrow Transplant. 2005
Jun;35(12):1127-32.
Owen RG, Hillmen P, Rawstron AC. CD52 expression in Waldenstrom's macroglobulinemia:
implications for alemtuzumab therapy and response assessment. Clin Lymphoma. 2005
Mar;5(4):278-81.
U
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Revision Nº 2
Emission date: 2013-02-13
HT-0052-CFB-1