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.

CD22 Antibodies

Mouse anti-human CD22 antibodies

-2 clones  

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1391clb              CLB-B-ly/1,6B11          III            mIgG1       purified    $375.00

RDI-M1437clb                " "                                 "                "             FITC       $375.00

RDI-M1727clb                " "                                 "                 "             PE          $438.00

RDI-CBL147                 RFB4                            III            mIgG1       purified    $375.00

RDI-CBL147FT               " "                                "                "             FITC       $375.00

RDI-CBL147PE               " "                                "                "             PE           $469.00

Product Specification: mouse monoclonal anti-Human CD22

PeliCluster  CD22

CAT#RDI- M1391clb    $375.00/vial

Test/vial 200

Clone CLB-B-ly/1, 6B11

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with cells of a patient with human Hairy Cell Leukaemia. This antibody has been clustered to CD22 in the third international Workshop on Human White Cell differentiation Antigens in Oxford (1986).

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 CD22- antigen, which is expressed on human B lymphocytes. Normal T-cells, polymorph nuclear cells, monocytes and platelets were found to be negative. Cell lines SB, Raji and NALM-1 were found to be positive. Most CALLA-ALL were found to be positive. The reaction pattern to CLL and NHL is variable from weak to negative. PLL is clearly positive and Hairy Cell Leukaemia is very strong positive with this antibody.

Molecular mass 130 kDa.

Application Enumeration of B lymphocytes in peripheral blood.

Identification of B lymphocytes in lymphoid tissue.

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

References 1. Bos, M.J.E. et al., McMichael et al. (editor), Leukocyte Typing III, Oxford University Press (1987).

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.

- microplate 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 microplate wells or tubes.

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

5 Incubate for 30 minutes at 2-8 C.

6 Add 100 µl buffer to the microplate 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 microplate 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: mouse monoclonal anti-CD22,conjugated to  Fluorescein.

PeliCluster  CD22 F

CAT#RDI-M1437clb   $375.00/vial

Test/vial 100

Clone CLB-B-ly/1, 6B11

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with cells of a patient with human Hairy Cell Leukaemia. This antibody has been clustered to CD22 in the third international Workshop on Human White Cell differentiation Antigens in Oxford (1986).

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 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 CD22- antigen, which is expressed on human B lymphocytes. Normal T-cells, polymorph nuclear cells, monocytes and platelets were found to be negative. Cell lines SB, Raji and NALM-1 were found to be positive. Most CALLA-ALL were found to be positive. The reaction pattern to CLL and NHL is variable from weak to negative. PLL is clearly positive and Hairy Cell Leukaemia is very strong positive with this antibody.

Molecular mass 130 kDa.

Application Enumeration of B lymphocytes in peripheral blood.

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

References 1. Bos, M.J.E. et al., McMichael et al. (editor), Leukocyte Typing III, Oxford University Press (1987).

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

- microplate 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 microplate wells or tubes.

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

4 Incubate for 30 minutes at 2-8°C.

5 Add 150 µl buffer to the microplate 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 microplate 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 test tubes, 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 lymphocyte 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

ANTI-HUMAN CD22 + B LYMPHOCYTES

PRODUCT CODE :RDI- CBL 147     also available $375.00/vial 200ug

     FITC labeled cat#RDI-CBL147FT       $375.00/vial 100 tests

        PE labeled cat#RDI-CBL147PE       $469.00/vial 100 tests

CLONE: RFB4

ISOTYPE: IgG1

SOURCE: Tissue culture supernatant

PURIFICATION METHOD: Protein A affinity chromatography

SPECIFICITY: Heterodimer composed of two glycoprotein chains of 130 - 140 kD molecular weight glycoprotein chains The antigen is present as both a cytoplasmic and membrane antigen on B lymphocytes.

ANTIGEN DISTRIBUTION: The CD22 antigen is expressed in the cytoplasm of the majority of B lymphocytes including early B progenitor cells. Antigen expression appears at an early stage in B lymphocyte differentiation at approximately the same stage as the CD19 antigen. Surface expression of the CD22 antigen by B lymphocytes is variable. Circulating B lymphocytes are CD22+ and follicular mantle and marginal zone B lymphocytes show strong binding of CBL147. Germinal centre B cells exhibit only weak expression of the CD22 antigen. The CD22 antigen is poorly expressed by non-T ALL cells and by a proportion of B- CLL cells. Hairy cell leukeamia cells have been demonstrated to strongly express the CD22 antigen.

B-cells (Ig+,E-) 92 ± 3%

Peripheral blood lymphocytes 12 ± 3%

Thymocytes < 1%

Granulocytes < 1%

Monocytes < 1%

T-cells (E+) < 1%

APPLICATIONS:

* Identification of B lymphocytes and HCL cells by indirect immunofluorescence staining

* Identification of B lymphocytes and B lymphocyte progenitor cells by staining of cytoplasmic CD22 antigen using the APAAP technique

Staining of B lymphocytes in frozen tissue sections by indirect immunoperoxidase staining

REFERENCES: Leucocyte Typing III, Oxford University Press. (1987).

                          Schwart-Albiez, R. et al. Int. Immunol. 3, 623-33 (1991).

PRESENTATION: Each monoclonal is delivered in a sealed vial at a concentration of not less than 200µg/2ml in phosphate buffered saline containing 10mM sodium azide and 1mg/ml of bovine serum albumin. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE: Purified monoclonal antibodies are generally stable for long periods at 4oC. However repeated warming to room temperature and re-cooling may result in a loss of activity and hence effective working titre. It is recommended that monoclonal antibodies are aliquotted upon receipt and then stored at -20C. After thawing they should then be used as a stock solution for up to 60 days. Dilute working solutions are best prepared on the day ofuse.

For research use only. Not supplied for use in human 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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