rev: September 9, 2004

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CD CLUSTERED ANTIBODIES  

(anti-Human and others as indicated)

NOTE: 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.


CD66E Antibodies


Mouse anti-human CD66E (CD66acde)  antibodies

-1 clone  


CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1545clb              CLB-gran/10                IV            mIgG1      purified     $375.00

RDI-M1643clb                    "                              "                 "            FITC        $375.00

RDI-M1693clb                    "                              "                 "            PE            $469.00


Product Specifications: mouse monoclonal anti-human CD66e

PeliCluster CD66e

cat#RDI-M1545clb

Test/vial 200

Clone CLB-gran/10, IH4Fc


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 CD66e 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 Affinity 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: 0.1% sodium azide


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 CD66e-antigen, which is expressed on mature human granulocytes. After granulocyte activation the expression is strongly increased. The monoclonal antibody reacts with 100% of mature human peripheral granulocytes. The monoclonal antibody reacts weak with malignant cells of patients with B-cell derived Chronic Lymphoid Leukaemia (CLL). The monoclonal antibody does not react with normal human peripheral B-cells, T-cells, monocytes and platelets. In immunohistology the monoclonal antibody reacts with some tissue macrophages and CEA, the Carcino Embryonic Antigen expressed on colon carcinoma and other carcinomas.


Molecular mass 180-200 kDa (phosphoprotein), 90 kDa (PI-linked protein).


Application Monitoring of mature granulocytes in peripheral blood.


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


References

1. Schoot, C.E. van der et al., Knapp, W. et al. (editors), Leucocyte Typing IV, 838 (1989).

2. Harvath, L. et al., Tissue Antigens, 33, 215 (1989).

3. Tetteroo, P.A.T. et al, J. Immunology, 136, 3427 (1986).


For research only!


Application  FOR AN INDIRECT IMMUNOFLUORESCENCE TECHNIQUE


Reagents

- FITC-conjugated goat anti-mouse immunoglobulin antiserum (M1204).

   Remove aggregates by centrifugation at 1000 x g for 10 minutes.

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

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

- Microtiter plates (96 wells, V bottom) or tubes.


Procedure

1  Fixate the cells with PFA 1% and wash them with buffer.

2  Prepare a mononuclear cell suspension with a concentration of 1 x 10 7 cells/ml in buffer.

3  Add 45 µl of cell suspension to the microtiter wells or tubes.

4  Add 5 µl monoclonal antibody to the microtiter wells or tubes and mix gently.

5  Incubate for 30 minutes at 2-8 C.

6  Add 100 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

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

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

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

10 Add 50 µl FITC-conjugated goat anti-mouse immunoglobulin serum, solid phase adsorbed with human immunoglobulins,      diluted 1:80.

11 Incubate for 30 minutes at 2-8 C.

12 Repeat step 6-9.

13 Add 200 µl buffer.

14 The number of positive cells can be determined by flowcytometry analysis or by means of fluorescence microscopy.


NOTE: Care should be taken when drawing blood to avoid activation of platelets.


Platelets activation markers are weakly expressed on platelets prepared from directly fixed normal whole blood.

The expression is maximal on thrombin (1 U/ml) stimulated washed platelets.

FOR IN VITRO RESEARCH USE ONLY


Product Specification: Murine monoclonal CD66,conjugated to Fluorescein


PeliCluster CD66e F


cat#RDI-M1643clb


Test/vial 100


Clone CLB-gran/10, IH4Fc


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 CD66e 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 Affinity 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 :0.1% sodium azide


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 CD66e-antigen, which is expressed on mature human granulocytes. After granulocyte activation the expression is strongly increased. The monoclonal antibody reacts with 100% of mature human peripheral granulocytes. The monoclonal antibody reacts weak with malignant cells of patients with B-cell derived Chronic Lymphoid Leukaemia (CLL). The monoclonal antibody does not react with normal human peripheral B-cells, T-cells, monocytes and platelets. In immunohistology the monoclonal antibody reacts with some tissue macrophages and CEA, the Carcino Embryonic Antigen expressed on colon carcinoma and other carcinomas.


Molecular mass 180-200 kDa (phosphoprotein), 90 kDa (PI-linked protein).


Application Monitoring of mature granulocytes in peripheral blood.


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


References

1. Schoot, C.E. van der et al., Knapp, W. et al. (editors), Leucocyte Typing IV, 838 (1989).

2. Harvath, L. et al., Tissue Antigens, 33, 215 (1989).

3. Tetteroo, P.A.T. et al, J. Immunology, 136, 3427 (1986).


Application in direct Immunofluorescence 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

- Microtiter 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 microtiter 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 immunoluorescently 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-Not for Use in Diagnostics


RDI Division of Fitzgerald Industries Intl

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

Ordering terms

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