rev: November 8, 1999

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CD CLUSTERED ANTIBODIES  

(anti-Human and others as indicated)

NOTE: 10% Discount on purchases of  3 or more any combination of CD antibodies. Please inquire for bulk or custom formulations (without preservative or carrier).

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (flow cytometry, neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below. All products are for in vitro research use only-not for use in or on humans or animals-not for use in diagnostics. Price/availability/specifications subject to change without notice.

CD66b Antibodies

-2 clone s 

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1546clb              CLB-B13.9                  IV            mIgG1      purified     $375.00

RDI-M1594clb                    "                              "                 "            FITC        $375.00

RDI-CD66b-G10F5       G10F5                         V              mIgM      purified     $375.00

 see sample protocols for conjugated antibody

PeliCluster CD66b

cat#RDI- M1546clb

Test/vial 200

Clone CLB-B13.9

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a (BALB/c x A/J) mouse immunized with human granulocytes. This antibody has been clustered to CD66b in the fifth international Workshop on Human White Cell differentiation Antigens in Boston (1993).

Isotype Mouse IgG1.

Source Ascites fluid of tumour bearing BALB/c mice.

Purification Ammoniumsulphate precipitation and ion exchange chromatography.

Packing Each vial contains 1 ml with approximately 0.2 mg/ml monoclonal antibody and 10 mg BSA in 20 mM TRIS and 150 mM NaCl, pH 8.0.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD66b- antigen, which is only expressed on the membrane of mature human neutrophil, eosinophil and basophil granulocytes. After granulocyte activation the expression is increased. The monoclonal antibody reacts with 100% of mature human granulocytes. The monoclonal antibody does not react with normal human peripheral B-cells, T-cells, monocytes and platelets.Molecular mass 100 kDa (PI-linked protein).

Application Monitoring of mature granulocytes in peripheral blood.

Analysis of peripheral blood granulocytes from patients with Paroxysmal Nocturnal Haemoglobinuria (PNH).

Methods Indirect immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

References 1. Schoot, C.E. van der, et al., Knapps, W. et al. (editors), Leucocyte Typing IV, 840 (1989).

FOR IN VITRO RESEARCH USE ONLY

PeliCluster   CD66b F

cat#RDI-M1594clb

Test/vial 100

Clone CLB-B13.9  This clone has been derived from hybridization of SP2/0 cells with spleen cells of a (BALB/c x A/J) mouse immunized with human granulocytes. This antibody has been clustered to CD66b in the fifth international Workshop on Human White Cell differentiation Antigens in Boston (1993).

Isotype Mouse IgG1.

Source Ascites fluid of tumour bearing BALB/c mice.

Purification Ammoniumsulphate precipitation and ion exchange chromatography.

Conjugation Cojugated with fluorescein iso thiocyanate isomer 1 (FITC).

Molecular F/P ratio between 5.5 - 9.5.

Packing Each vial contains 1 ml FITC conjugated monoclonal antibody and 10 mg BSA in PBS.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD66b- antigen, which is only expressed on the membrane of mature human neutrophil, eosinophil and basophil granulocytes. After granulocyte activation the expression is increased. The monoclonal antibody reacts with 100% of mature human granulocytes. The monoclonal antibody does not react with normal human peripheral B-cells, T-cells, monocytes and platelets.Molecular mass 100 kDa (PI-linked protein).

Application Monitoring of mature granulocytes in peripheral blood.

Analysis of peripheral blood granulocytes from patients with Paroxysmal Nocturnal Haemoglobinuria (PNH).

Methods Direct immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

References 1. Schoot, C.E. van der, et al., Knapps, W. et al. (editors), Leucocyte Typing IV, 840 (1989).

For research only!

APPLICATION in directImmunofluorescence techniques

Method with ficoll purified cells

Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumine (BSA)

- PFA: Para-Formaldehyde 1%, pH 7.2,  containing 0.2% BSA

- Microwell plates (96 wells, V bottom) or tybes.

Procedure

1   Prepare a mononuclear cell suspension with a concentratation of 1 x 10 7 cells/ml.

2   Ad 40 µl of cell suspension to microtiter wells or tubes.

3   Add 10 µl of the antibody to the microtiter wells or tubes and mix gently.

4   Incubate for 30 minutes at 2-8°C.

5   Add 150 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

6   Aspirate the supernatant from the cell pellet and resuspend the cells.

7   Add 200 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

8   Aspirate the supernatant from the cell pellet and resuspend the cells.

9   Cytoflow fluorometer analysis:

     Add 200 µl buffer to the microplate wells and transfer this final cell suspension to appropriate testtubes, or add 200 µl            buffer to the tubes.

10  If analysis within 8 hours is not possible add at no. 9, instead of buffer, 200 µl PFA 1%.

Fig. 1

Fluorescense profile of ficoll purified normal cells

(Scatter gates set on the granulocyte fraction)

WHOLE BLOOD METHOD

Due to sophisticated laser flowcytometry we are now able to distinguish populations of human lymphocytes, monocytes and granulocytes by their characteristics in different light scattering. By gating on the appropriate cell cluster (Fig.2) we can easily determine mononuclear cells, which have been immunofluorescently stained with monoclonal antibodies, and enumerate them.

Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Lysing solution (NH 4Cl, pH 7.2).

- Buffer: PBS, containing 0.2 % BSA.

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2 % BSA.

- Tubes.

Procedure

1  Draw blood into a blood collection tube containing EDTA.

2  Deliver 100 µl (*) of well mixed whole blood to the bottom of the testtube.

3  Add 10 µl of the undiluted antibody to the bottom of the testtube, and mix gently.

4  Incubate for 30 minutes at 2-8°C or in an ice-water bath.

5  Mix the tubes and add 2 ml of lysing solution.

6  Incubate for 3-5 minutes at room temperature.

7 When lysing is completed, centrifuge the tubes at 500 x g for 5 minutes.

8  Aspirate the supernatant from the cell pellet, resuspend the cells in 1 ml buffer.

    Place the tubes in the ice-water bath.

9  Analyse the samples within 8 hours.

If analysis within 8 hours is not possible centrifuge the tubes at 500 x g. Aspirate the supernatant from the cell pellet and resuspend the cells in 1 ml PFA 1%.

Fig. 2:

Scatter patern of a whole normal blood sample

R2 - lymphocytes R3 - monocytes R4 - granulocytes

Note:Use our special negative control to determine background fluorescence produced due to Fc binding capacities by mononuclear cells.The concentration and F/P ratio of our control has been adjusted to our FITC-conjugated monoclonal antibodies.* This method was developed for blood samples with a normal white count. It may be necessary to adjust the quantity of blood for samples with very high or low white counts

FOR IN VITRO RESEARCH USE ONLY

Catalog#: RDI-CD66B-G10F5   $281.00/vial

Package Size: 100ug purified in 02.ml (0.5mg/ml) 10mM sodium phosphate pH7.2, 500mM NaCl and 0.09% sodium azide. (sodium azide is toxic,dilute azide compounds with copious amounts of water when disposing in drain to avoid accumulation of explosive deposits-avoid contact with acids.)

Prep: purified from tissue culture supernatant via Protein G affinity chromatography.

Species: mouse IgMk

CLONE: G10F5

Workshop Code: Workshop V, MA020

Activity: Reacts with a 100Kd, a glycosylphosphatidylinositol (GPI) anchored protein expressed on granulocytes. This molecule was previously clustered as CD67 in the IVth Workshop amd renamed CD66B in the Vth HLDA Workshop. CD66b is a member of the CEA like glycoprotein family present on granulocytes and referred to as non specific cross reacting antigenss. Granulocyte activation induced with soluble stimulators (calcium ionophore, phorbol myristate acetate, N- forylmethionyl-leucyl-phenylalanine)results in release and increased expression of NCA. Findings suggest that these molecules may play a role in phagocytosis, chemotaxis and adherence.

Application: -Indirect immunofluorescence cell surface staining , Immunohistochemistry on formalin-fixed paraffin embedded tisue sections using citrate pretreatment and ABC/Dab detection. Recommend using a specific anti-IgM secondary antibody.Titer for each application

References: 1) Schlossman, S. et al, ed.s 1995, Leucocyte Typing V, White Cell Differentiation Antigens, Oxford University Press, NY

                   2) Eur J. Immunol. 23:2782 (1993)

                   3) J. Immunol. 151:4934 (1993)

                   4) Journal of Leukocyte Biology 52:551.(1992)

Storage: Store at 4 DEG C.

Precautions:For In vitro research Use Only. Not for use in or on humans or animals or or diagnostics.

Fitc labeled cat#RDI-CD66B-G10-FT $469.00/100 T vial

It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics.

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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