rev:July 23, 1999

HOME (index  HOME page)

Return  (master CD antibody index page)

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.

CD24 Antibodies

Mouse anti-human CD24 antibodies

-2 clones  

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1361clb              CLB-gran-B-ly/1,1B5    III             mIgG1     purified      $375.00

RDI-M1605clb                  " "                                "                "           FITC         $375.00

RDI-CBL561                 SN3                                 IV            mIgG1     purified      $438.00

PeliCluster  CD24

CAT#RDI- M1361clb

Test/vial 200

Clone CLB-gran-B-Ly/1, 1B5

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human granulocytes. This antibody has been clustered to CD24 in the third international Workshop on Human White Cell differentiation Antigens

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 CD24- antigen, which is expressed on virtually all B-cells: early B-cell precursors, pre-pre-B-cells, pre-B-cells, B- cells, intermediate B-cells, mature B-cells and some plasmacytoid cells are positive while plasmacells are negative. The monoclonal antibody reacts with human granulocytes and their precursors (from promyelocytic stage). The monoclonal antibody also reacts with virtually all pre-B-cell lines and Burkitt cell lines, but is only expressed on less then 50% of the B-cell lymphoblastoid cell lines. Non T-ALL, B-cell NHL and 50% of myelomas as well as a subpopulation of AML is found to be positive. Hairy Cell Leukaemia is found to be weakly positive.

Molecular mass 45, 55, 65 kDa.

Application Identification of B-CLL..

Immunophenotyping of Hairy Cell Leukaemia.

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

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

PeliCluster  CD24 F

CAT#RDI-M1605clb

Test/vial 100

Clone CLB-gran-B-Ly/1, 1B5

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human granulocytes. This antibody has been clustered to CD24 in the third international Workshop on Human White Cell differentiation Antigens

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 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 CD24- antigen, which is expressed on virtually all B-cells: early B-cell precursors, pre-pre-B-cells, pre-B-cells, B- cells, intermediate B-cells, mature B-cells and some plasmacytoid cells are positive while plasmacells are negative. The monoclonal antibody reacts with human granulocytes and their precursors (from promyelocytic stage). The monoclonal antibody also reacts with virtually all pre-B-cell lines and Burkitt cell lines, but is only expressed on less then 50% of the B-cell lymphoblastoid cell lines. Non T-ALL, B-cell NHL and 50% of myelomas as well as a subpopulation of AML is found to be positive. Hairy Cell Leukaemia is found to be weakly positive.

Molecular mass 45, 55, 65 kDa.

Application Identification of B-CLL..

Immunophenotyping of Hairy Cell Leukaemia.

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

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 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

PRODUCT CODE    RDI-CBL561    $438.00/vial

CLONE   SN3

ISOTYPE   IgG1

SOURCE   Tissue culture supernatant

SPECIFICITY

The antibody reacts with the CD24 antigen a heavily glycosylated molecule that migrates as a broad band of 35-45 kDa on both reducing and non-reducing SDS gel electrophoresis. CD24 is attached to the cell membrane via a glycosyl phosphatidylinositol (GPI) anchor and is expressed at multiple stages of B cell development, beginning with the bone marrow CD34 positive pro-B cell compartment and continuing through mature surface IgM positive/IgD positive B cells. CD24 is also expressed on the vast majority of B-lineage acute lymphoblastic leukaemias, B cell chronic lymphocytic leukaemias and B cell non-Hodgkin's lymphoma. In the normal spleen, mantle zone lymphocytes are CD24 strongly positive, but germinal center cells are CD24 negative or CD24 weakly positive.

APPLICATIONS

*Immunocytochemistry on frozen tissue sections.

*FACS analysis of CD24

REFERENCES

Pirruccello, S.J. LeBien, T.W. J. Immunol. 136, 3779-84 (1986)

Fischer, G.F. et al. J. Immunol. 144, 638-41 (1990)

Solvason, N. Kearney, J.F. J. Exp. Med. 175, 397-404 (1992)

Kersey J, et al. Lancet 2, 1419-23 (1982)

Fukukawa, T. et al. Exp. Hamatol 14, 850-5 (1986)

Perri, R.T. et al. Blood 51, 871-5 (1983)

Lebien T, et al. Leuk. Res. 6, 229-306 (1962)

Leucocyte Typing IV Oxford University Press (1989)

Barcos, M. et al. Hematological Oncol. 4, 251-9 (1986)

PRESENTATION

The monoclonal is presented at a concentration of 200µg/2ml in phosphate buffered saline containing 10mM sodium azide and 1mg/ml bovine serum albumin. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE For use within 1 month of purchase store at +4oC, for long term storage aliquot antibody into small volumes and store at -20oC.

Bulk material quoted upon request (minimum 1 mg) approx $1125.00/mg available without BSA or preservative Larger sizes quoted upon request

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

RETURN (CD Antibody INDEX page)