rev:  August 11, 2001

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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.

CD33 Antibodies

Mouse anti-human CD33 antibodies

-2 clones  

CATALOG#                 CLONE#                  Wokshop    Host         Form        Price

RDI-M1585clb              CLB-MD33.6                 --           mIgG1       purified     $375.00

RDI-M1606clb                   " "                               "               "             FITC       $375.00

RDI-M1662clb                  " "                                "              "              PE           $469.00

RDI-CBL163                 WM53                           IV          mIgG1        purified    $438.00

RDI-CBL163FT                " "                                "              "              FITC       $438.00

RDI-CBL163PE                " "                                "              "              PE             $500.00

RDI-CBL163PC5             "  "                                "              "              RPE-CY5  $500.00

RDI-013-1     CD33 purified       $375.00/100ug      $656.00/250ug      $2094.00/1 milligram

RDI-013-4     CD33 FITC           $500.00/100ug       $938.00/250ug     $3344.00/1 milligram

-reacts with a 67 kDa polypeptide expressed on hematopoetic progenitor cells, monocytes, and  acute myeloid leukemia (AML ) cells from the majority of patients.

PeliCluster  CD33

CAT#RDI-M1585clb

Test/vial 200

Clone CLB-MD33.6    This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human CD33 transfectants. This antibody has been clustered to CD33 in one of the 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 CD33- antigen (belonging to the Ig-supergene family), which is expressed on human myelomonocytic cells. The monoclonal antibody reacts in the bone marrow from myeloblasts up to myelocytes. CD33-antigen is found on CFU-GEMM, CFU-GM, CFU-G, CFU-M and with erythroid CFU-E but not on earlier precursors. The monoclonal antibody does not react with normal human peripheral granulocytes, B-cells, T-cells and platelets. The monoclonal antibody reacts weakly with blast cells in 70% of patients with Acute Myeloid Leukaemia (AML) and in 30% of adult patients with Acute Lymphoblastic Leukaemia (ALL).

Molecular mass 67 kDa.

Application Analysis of myeloid leukaemia and studies of myeloid differentiation.

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

APPLICATION  FOR AN INDIRECT IMMUNOFLUORESCENCE TECHNIQUEReagents

- 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.

- microwell 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 microwell wells or tubes.

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

5 Incubate for 30 minutes at 2-8 C.

6 Add 100 µl buffer to the microwell 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 microwell 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 CD33 F     CAT#RDI- M1606clb

Test/vial 100

Clone CLB-MD33.6   This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human CD33 transfectants. This antibody has been clustered to CD33 in one of the 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 Conjugated 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 CD33- antigen (belonging to the Ig-supergene family), which is expressed on human myelomonocytic cells. The monoclonal antibody reacts in the bone marrow from myeloblasts up to myelocytes. CD33-antigen is found on CFU-GEMM, CFU-GM, CFU-G, CFU-M and with erythroid CFU-E but not on earlier precursors. The monoclonal antibody does not react with normal human peripheral granulocytes, B-cells, T-cells and platelets. The monoclonal antibody reacts weakly with blast cells in 70% of patients with Acute Myeloid Leukaemia (AML) and in 30% of adult patients with Acute Lymphoblastic Leukaemia (ALL).

Molecular mass 67 kDa.

Application Analysis of myeloid leukaemia and studies of myeloid differentiation.

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

- 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 microwell wells or tubes.

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

4 Incubate for 30 minutes at 2-8°C.

5 Add 150 µl buffer to the microwell 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 microwell 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 microwell 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 monocyte 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

PeliClusterCD33 PE

CAT#RDI-M1662clb

Test/vial 100

Clone CLB-MD33.6  This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human CD33 transfectants. This antibody has been clustered to CD33 in one of the 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 Conjugated with R-phycoerythrin (PE).

Molecular F/P ratio between 1.0 - 2.0.

Packing Each vial contains 1 ml PE conjugated monoclonal antibody and 10 mg BSA in 20 mM TRIS and 150mM 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 CD33- antigen (belonging to the Ig-supergene family), which is expressed on human myelomonocytic cells. The monoclonal antibody reacts in the bone marrow from myeloblasts up to myelocytes. CD33-antigen is found on CFU-GEMM, CFU-GM, CFU-G, CFU-M and with erythroid CFU-E but not on earlier precursors. The monoclonal antibody does not react with normal human peripheral granulocytes, B-cells, T-cells and platelets. The monoclonal antibody reacts weakly with blast cells in 70% of patients with Acute Myeloid Leukaemia (AML) and in 30% of adult patients with Acute Lymphoblastic Leukaemia (ALL).

Molecular mass 67 kDa.

Application Analysis of myeloid leukaemia and studies of myeloid differentiation.

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

- 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 microwell wells or tubes.

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

4 Incubate for 30 minutes at 2-8°C.

5 Add 150 µl buffer to the microwell 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 microwell 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 microwell 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 monocyte 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 M1662/02 0708961415

 PRODUCT CODE :RDI-CBL-163     $438.00/vial 200ug purified (min 200 tests)

Also available: FITC labeled cat#RDI-CBL163FT    $438.00/vial 100 tests

                           PE labeled cat#RDI-CBL163PE    $500.00/vial 100 tests

                       RPE-CY5 labeled cat#RDI-CBL163PC5 $500.00 100 tests

MOUSE MONOCLONAL  ANTIBODY TO: HUMAN CD33

PRODUCT CODE   RDI-CBL163    $438.00/vial

CLONE    WM53

ISOTYPE    IgG1

SOURCE     Mouse ascitic fluid

PURIFICATION METHOD

Ion exchange chromatography

SPECIFICITY   The antigen is a single chain transmembrane glycoprotein of molecular mass 67 kDa expressed on monocytes, myeloid progenitor cells and myeloid leukaemias. The antigen has homology with CD22 and myelin associated glycoprotein.

APPLICATIONS

*Phenotyping of leukaemias, useful in distinguishing myelogenous leukaemias from lymphoid or erythroid leukaemias

*Bone marrow purging in autologous bone marrow transplantation

REFERENCES

Leucocyte Typing III, Oxford University Press (1987)

Leucocyte Typing IV, Oxford University Press (1989)

Favaloro, E. et al. Br. J. Haem. 69, 163-71 (1986)

Favaloro, E. J. Immunol. & Cell Biol. 71, 571081 (1993)

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.

Also available: FITC labeled cat#RDI-CBL163FT $438.00/100T

PE labeled cat#RDI-CBL163PE $500.00/100T

RPE-CY5 labeled cat#RDI-CBL163PC5 $500.00/100T

Bulk , purified (NO BSA)      cat#RDI-CBL163-1X     $1188.00/1mg

NO AZIDE/NO BSA             cat#RDI-CBL163-1XP   $1188.00/1mg

Larger sizes quoted on request

For Research Use Only. Not supplied for use in diagnostic or therapeutic procedures

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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