CD11a Antibodies

CD11a Antibodies

Mouse anti-human CD11a antibodies

-2 clones     CLB-LFA 1/2.TB133 (purified & FITC)

                    38 (purified, FITC & PE)

CATALOG#                 CLONE#                  Wokshop  Host         Form        Price

RDI-M1385clb               CLB-LFA1/2,TB133    IV           mIgG2a    purified     $375.00

RDI-M1667clb                " "                                "                 "           FITC        $375.00

RDI-CBL451                  38                                --             mIgG2a    purified     $375.00

RDI-CBL451FT              " "                               --                "            FITC        $375.00

RDI-CBL451PE              " "                               --                "            PE            $469.00

Product Specification: mouse monoclonal anti-human CD11a

PeliCluster CD11a

cat#RDI- M1385clb

Test/vial 200

Clone CLB-LFA-1/2, TB-133

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with activated T-cells. This antibody has been clustered to CD11a in one of the international Workshop on Human White Cell differentiation Antigens

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 0.1 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 CD11a- antigen, located on the alpha-L chain of LFA-1 complex (Lymphocyte Function-associated Antigen-1), which is expressed on mature immunocompetent lymphocytes and their neoplastic counterparts, granulocytes and monocytes.

The monoclonal antibody does not react with human thrombocytes.

Molecular mass 170 kDa.

Application The monoclonal antibody can be used to detect LFA-1 deficiency in patients suffering from recurrent bacterial infections.

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

mouse anti-human CD11a:FITC conjugated

PeliCluster  CD11a F

CAT#RDI-M1667clb

Test/vial 100

Clone CLB-LFA-1/2, TB-133

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with activated T-cells. This antibody has been clustered to CD11a in one of the international Workshop on Human White Cell differentiation Antigens

Isotype Mouse, IgG2a.

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 6.0 - 10.0.

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 CD11a- antigen, located on the alpha-L chain of LFA-1 complex (Lymphocyte Function-associated Antigen-1), which is expressed on mature immunocompetent lymphocytes and their neoplastic counterparts, granulocytes and monocytes.

The monoclonal antibody does not react with human thrombocytes.

Molecular mass 170 kDa.

Application The monoclonal antibody can be used to detect LFA-1 deficiency in patients suffering from recurrent bacterial infections.

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 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-NOT FOR USE IN DIAGNOSTICS

ANTI-HUMAN CD11a ANTIGEN

PRODUCT CODE :RDI-CBL451    $375.00/vial 200ug

cat#RDI-CBL451FT    $375.00/vial     100 tests FITC labeled

     #RDI-CBL451PE    $469.00/vial     100 tests PE labeled


CLONE: 38

ISOTYPE: IgG2a

SOURCE: Tissue Culture supernatant

Fusion Partner: SP2/0 myeloma cell line

PURIFICATION METHOD: Protein A affinity chromatography.

SPECIFICITY: Reacts strongly with all leucocytes in both histological and flow cytometric studies and recognizes the 180kDa a-chain of the leucocyte function antigen - I (LFA-I) which is a heterodimeric glycoprotein. As a member of the integrin family (a-subunit) the antigen enables adhesion of lymphoid cells to the vascular endothelium in association with its ligand ICAM-1.

Antigen Distribution: Thymocytes > 90%

                               Granulocytes > 95%

                               Monocytes > 95%

                               LGL cells > 95%

                               B-cells (CD20+) > 95%

                               T-cells (CD3+) 95%

                               Peripheral blood lymphocytes > 95%

APPLICATIONS:

* Characterisation of LFA-I immunodeficiency.

* Studies involving ICAM-I binding, cell-cell adhesion and cell mediated cytotoxicity.

* Studies of the CD11a moiety in both flow cytometry and on frozen tissue sections

* Studies on adhesion blockade of the CD11a antigen


REFERENCES: Dransfield, I. & Hogg, N. EMBO J. 12, 3759 (1989).

PRESENTATION: Each monoclonal is delivered in a sealed vial at a concentration of 200µg/2ml in phosphate buffered saline solution containing 10mM sodium azide& 1mg/ml BSA.. 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 of use.