rev: April 7, 2000

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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.

CD15 & CD15s Antibodies

-2 clones     CLB-gran/2,B4  (purified & FITC)

                    28  (purified, FITC & PE)

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1336clb              CLB-gran/2, B4              IV           mIgM       purified      $375.00

RDI-M1435clb                " "                                 "                "             FITC         $375.00

RDI-CBL144                  28                                 IV           mIgM       purified      $375.00

RDI-CBL144FT               " "                                "                "             FITC         $375.00

RDI-CBL144PE               " "                                "                "             PE             $469.00

PeliCluster  CD15   cat#RDI- M1336clb   $375.00/vial

Test/vial 200

Clone CLB-gran/2,B4  This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c x A/J mouse immunized with Fc-gamma positive T lymphocytes. This antibody has been clustered to CD15 in one of the international Workshop on Human White Cell differentiation Antigens

Isotype Mouse, IgM

Source Hybridoma supernatant.

Purification Ammoniumsulphate precipitation and gelfiltration.

Packing Each vial contains 1 ml with approximately 0.1 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 CD15- antigen (the FAL structure) of human polymorphonuclear cells. The monoclonal antibody reacts with the promyelocytes, myelocytes and polymorphonuclear cells. After neuramidase treatment of cells the FAL structure is expressed on all cells of the monocytic and myelocytic lineage. This monoclonal antibody does not react with platelets and cells of the T and B lymphocyte lineage.

Molecular mass 105, 150 kDa.

Application Analysis of myeloid leukaemias and studies of myeloid differentiation.

Identification of Reed-Sternberg cells in Hodgkin's disease.

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

References

1. Tetteroo, P.A.T., Mulder, A., Lansdorp, P.M., Zola, H., Baker, D.A., Visser, F.J., Borne, A.E.G.Kr. von dem, Eur. J. of Immunol., 14, 1089-1095 (1984).

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

PeliCluster  CD15 F    cat#RDI- M1435clb    $375.00/vial

Test/vial 100

Clone CLB-gran/2,B4   This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c x A/J mouse immunized with Fc-gamma positive T lymphocytes. This antibody has been clustered to CD15 in one of the international Workshop on Human White Cell differentiation Antigens

Isotype Mouse, IgM

Source Hybridoma supernatant.

Purification Ammoniumsulphate precipitation and gelfiltration.

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

Molecular F/P ratio between 18 - 30.

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 CD15- antigen (the FAL structure) of human polymorphonuclear cells. The monoclonal antibody reacts with the promyelocytes, myelocytes and polymorphonuclear cells. After neuramidase treatment of cells the FAL structure is expressed on all cells of the monocytic and myelocytic lineage. This monoclonal antibody does not react with platelets and cells of the T and B lymphocyte lineage.

Molecular mass 105, 150 kDa.

Application Analysis of myeloid leukaemias and studies of myeloid differentiation.

Identification of Reed-Sternberg cells in Hodgkin's disease.

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

References

1. Tetteroo, P.A.T., Mulder, A., Lansdorp, P.M., Zola, H., Baker, D.A., Visser, F.J., Borne, A.E.G.Kr. von dem, Eur. J. of Immunol., 14, 1089-1095 (1984).

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 mononucleair 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 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: Phosphate Buffered Saline (PBS), containing 0.2 % Bovine Serum Albumin (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 - lynphocytes 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-CBL144   $375.00/vial 200ug

Also available: FITC labeled cat#RDI-CBL144FT $375.00/100 Tests

                           PE labeled cat#RDI-CBL144PE $469.00/100 Tests

CLONE    28

ISOTYPE   IgM

IMMUNIZING ANTIGEN    Human monocytes

FUSION PARTNER    SP 2/0-Ag 14 myeloma cell line

SOURCE   Tissue culture supernatant

PURIFICATION METHOD   Ammonium sulphate precipitation followed by gel filtration

SPECIFICITY   The CD15 antigen is the X-hapten on lacto-N-fucose pentaosyl lll, which is expressed on circulating granulocytes or tissue granulocytes as well as neutrophils and eosinophils.

Antigen distribution:

   Granulocytes > 95%

   Monocytes 88 + 7%

  B  cells (E-) < 1%

  T cells (E+) < 1%

  Peripheral blood lymphocytes < 1%

APPLICATIONS

*Identification of CD15+ granulocytes by indirect immunofluorescence or by FACS analysis

*Identification of CD15+ granulocytes present in frozen sections

*Phenotyping of lymphomas and leukaemias

*Inhibition of neutrophil phagocytosis and bactericidal activity

*Precipitation studies of the CD15 antigen

REFERENCES

Leucocyte Typing Ill Oxford University Press (1987)

Hogg, N. Immunol. 53 753 (1984)

Stocks, S.C. et. al. Biochem. J. 268, 275-80 (1990)

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 +4°C, for long term storage aliquot antibody into small volumes and store at +4°C or add an equal volume of glycerol and store at -20°C.

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

Bulk material available without BSA or preservative on request, approx $1062.00/1 milligram, ord cat#RDI-CBL144-1XP. Larger discounts available-please inquire.

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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