rev: October 6, 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 monoclonal 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.

CD14 Antibodies

Mouse anti-human CD14 antibodies

-3 clones     CLB-mon/1(301),8G3  (purified, FITC & PE)

                   UCHM1 (purified, FITC & PE)

                   Chris 6 (purified)

CATALOG#                 CLONE#                  Wokshop    Host         Form        Price

RDI-M1388clb              CLB-mon/1(301),8G3   III & IV    mIgG2a    purified      $375.00

RDI-M1430clb               " "                                   "                "            FITC         $375.00

RDI-M1661clb               " "                                   "                "            PE             $469.00

RDI-CBL453                 UCHM1                         IV            mIgG2a   purified       $375.00

RDI-CBL453FT              " "                                  "                "            FITC         $375.00

RDI-CBL453PE              " "                                   "                "            PE            $469.00

RDI-CD14-CRIS6        CRIS 6                          III & IV    mIgG2a    purified     $406.00/100ug

PeliCluster  CD14

cat#RDI- M1388clb

Test/vial 200

Clone CLB-mon/1, 8G
This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human monocytes. This antibody has been clustered to CD14 in the third International Workshop on Human White Cell differentiation Antigens in Oxford (1986).

Isotype Mouse, IgG2a.

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 CD14- antigen, which is expressed on human monocytes and macrophages. The antibody reacts with human monocytes and macrophages; weak reactions may occur with neutrophils.

Molecular mass 55 kDa.

Application Enumeration of monocytes and macrophages in peripheral blood.

Identification of monocytes and macrophages in lymphoid tissues.

This monoclonal antibody is a useful marker for monocytic and myelomonocytic acute myeloid 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-NOT FOR USE In DIAGNOSTIC Procedures.

Product Specification: mouse monoclonal anti-human CD14 conjugated to FITC

PeliCluster CD14 F

cat#RDI-M1430clb

Test/vial 100

Clone CLB-mon/1, 8G3
This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human monocytes. This antibody has been clustered to CD14 in the third International Workshop on Human White Cell differentiation Antigens in Oxford (1986).

Isotype Mouse, IgG2a.

Source Ascites fluid of tumour bearing BALB/c mice.

Purification Ammoniumsulphate precipitation and ion exchange chromatography.

Conjugation Conjugated with fluorescein iso tiocyanate 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 CD14- antigen, which is expressed on human monocytes and macrophages. The antibody reacts with human monocytes and macrophages; weak reactions may occur with neutrophils.

Molecular mass 55 kDa.

Application Enumeration of monocytes and macrophages in peripheral blood.

Identification of monocytes and macrophages in lymphoid tissues.

This monoclonal antibody is a useful marker for monocytic and myelomonocytic acute myeloid 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 mononucleair 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 monocyte fraction)

Whole  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-Not For Use in Diagnostics

Test/vial 100

Clone CLB-mon/1, 8G3
This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human monocytes. This antibody has been clustered to CD14 in the third International Workshop on Human White Cell differentiation Antigens in Oxford (1986).

Isotype Mouse, IgG2a.

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 20mM TRIS and 150 mM NaCl, pH8.0.

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 CD14- antigen, which is expressed on human monocytes and macrophages. The antibody reacts with human monocytes and macrophages; weak reactions may occur with neutrophils.

Molecular mass 55 kDa.

Application Enumeration of monocytes and macrophages in peripheral blood.

Identification of monocytes and macrophages in lymphoid tissues.

This monoclonal antibody is a useful marker for monocytic and myelomonocytic acute myeloid 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

- PE-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 mononucleair cell suspension with a concentratation of 1 x 10 7 cells/ml.

2 Add 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 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 immunfluorescently 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

ANTI -CD14 ANTIGEN

PRODUCT CODE : RDI- CBL 453   $375.00/vial 200ug

       FITC labeled ord cat#RDI-CBL453FT $375.00/vial 100 tests

       PE labeled ord cat#RDI-CBL453PE $469.00/vial 100 tests

CLONE: UCHM-1

ISOTYPE: IgG2a

SOURCE: Mouse ascitic fluid.

PURIFICATION METHOD: Protein A affinity chromatography.

SPECIFICITY: 55kD MW monocyte surface antigen identified by monoclonal antibodies belonging to the CD14 cluster and found on peripheral blood monocytes and weakly on granulocytes. Monocytic cells, interfollicular dendritic cells and dendritic reticulum cells are stained in sections of lymphoid tissue sections.

Antigen distribution: Monocytes > 98%

                               Granulocytes > 60%

                                LGL-cells < 5%

                                Peripheral blood lymphocytes < 1%

                                T-cells (CD3+) < 1%

                                 B-cells (CD20+) < 1%

                                 Thymocytes < 1%

APPLICATIONS:

* Identification of monocytic cells by indirect immunofluorescence staining.

* Identification of cells of monocyte lineage by immunohistological staining of tissue sections.

* Study of follicular architecture in lymphoid tissue with malignant involvement.

* Immunoprecipitation of the CD14 antigen.

REFERENCES: Hogg, N. et al. Immunology 53, 753 (1984)

Linch et al. Blood 63, 566 (1984)

PRESENTATION: Each monoclonal is delivered in a sealed vial at a concentration of 200µg/2ml in phosphate buffered saline solution containing 10mM sodium azide. 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 -200C. 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 of use.

For research use only. Not supplied for use in human diagnostic or therapeutic procedures

Bulk (order cat#RDI-CBL453-1XP   $1125.00/milligram no BSA, no Preservative) Larger sizes on request

CD14 clone CRIS6 cat#RDI-CD14-CRIS6  $406.00/100ug   Larger sizes quoted on request

Package Size: 100ug purified ab in 0.1ml (1mg/ml)   PBS no carrier/no preservative

Species: mouse IgG1

CLONE: CRIS-6

Immunogen: Human peripheral blood mononuclear cells

Purification: Protein G of ascites

Activity: Reacts with human CD14. Recognizes a 55kda glycoprotein found on monocytes, macrophages, langerhans cells and neutrophils. It will immunoprecipitate a 69kda protein from PMA-stimulated U937 cells which is though to be the precursor to the 55kda glycoprotein.

Application: -flow cytometry (use approx 0.1-1ug to label 1 million cells)

                      -cryostat (frozen) sections

                     -immunoprecipitation

Storage: Store at -20 DEG C. Avoid multiple freeze/thaw cycles. Further dilution should be made in medium or bufered solution with carrier protein and preservative such as PBS with 1% BSA and 0.1% sodium azide.

ref: Leukocyte Typing IV (1989) Knapp, W et al eds p789.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics.  It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

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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