HLA Class 1 Antigen Bw4 (HLA-C) Antibody – Mouse Monoclonal

Antibodies can also be used for the detection of HLA antigens on the lymphocytes in whole blood samples and cells isolated from blood or lymphoid tissue. The HLA Class 1 conjugated monoclonal antibodies can be used for the detection of specific cell surface antigens.

Intended Use
These reagents are used for direct immunofluorescence staining of specific cell surface antigens with analysis by flow cytometry . Antibodies can also be used for the detection of HLA antigens on the lymphocytes in whole blood samples and cells isolated from blood or lymphoid tissue.

Summary and Explanation
The HLA Class 1 conjugated monoclonal antibodies can be used for the detection of specific cell surface antigens.

Principle
Conjugated monoclonal antibodies(Mabs) detect cells bearing the cell surface antigens specific to the antibody. Cells (whole blood or isolatea cells) are stained with the conjugated Mab, red blood cells are lysed and white blood cells are fixed with formaldehyde. Flow cytometric anlysis is performed on the target cells.

Identification
HLA Antigen Specific FITC Antibody Concentration: 0.1 mg/ml-O.5mg/ml Total volume: 250ul sufficient for 25 tests. F:P Ratio: 4.0-8.0

Note: All blood products should be treated as potentially infectious. Source material from which this product was derived was found negative when tested in accordance with current FDA required tests. No known test methods can offer assurance that products denved from known human blood will not trasmit infectious agents.

Treatment Required for Use
1. To avoid volume loss, centrifuge conjugated Mab vial for a few seconds in a microcentrifuge before opening (liquid may accumulate in cap during shipment).
2.vial is susceptible to volume loss if left uncovered. Cover vial immediately after use.

Instability Indications
Do not use the reagent if precipitate is observed.

Specimen Colledion and Preparation
1. The blood specimen should be collected in an ACD (Acid Citrate Dextrose) Vacutainer blood collection tube and analyzed within three days. However, EDTA (K3) or Sodium Heparin may also be used.
2. Sterile blood samples should be stored horizontally at room temperature (20°C) and analyzed within 3 days of collection.

Procedure
A. Materials Provided
1. FITC Conjugated Monoclonal Antibody. Warning: These reagents may contain sodium azide in the range of 0.1-1 % which under acidic conditions yields hydrazonic acid, an extremelY toxic compound. Reagents containing sodium azide snould be diluted in running water prior to being discarded. These conditions are recommended to avoid deposits in plumbing where explosive conditions may develop. See European Directive 67/548/EEC (Xn Harmful).

B. Materials Required, But Not Provided
1. Red Blood Cell lysing solution
2. Streptavidin-PE
3. Phosphate Buffered Saline (PBS) or equivalent
4. Fixing solution: PBS with 0.5% formaldehyde (add 1.35ml 37% formaldehyde to 100ml PBS).
Warning: Formaldehyde is toxic and allergenic, and is a suspected carcinogen. Formaldehyde is listed as a carcinogen in California. Avoid contact with eyes, skin and clothing.

Diredions for Use
A. Whole Blood Staining Method To avoid volume loss, centrifuge vial for a few seconds in a microcentrifuge before use (liquid may accumulate in cap during shipment).

1. Pipette 1 OOul of whole blood sample into a 12x75mm tube.
2. Pipette 10ul of H6098-61 A into the tube and mix well with gentle vortex.
Caution: Susceptible to volume loss if vial is left uncovered. Cover vial immediately ater use.
3. Incubate in the dark at 4°C for 30 minutes while gently shaking.
4. Lyse Red Blood Cells.
5. Centrifuge samples at 300 x g for 5 minutes. Discard the supernatant.
6. Resuspend pellet in 2ml of PBS, mix, and centrifuge at 300 x g for 5 minutes. Discard the supernatant. 7. For FITC conjugated Mab! go to step #9.
8. Repeat steps 5 and 6 twice.
9. Resuspend the pellet in 0.5 ml of fixing solution. The cells are ready for immediate flow cytometry analysis, or can be stored in the
dark at 4°C for up to 24 hours before being analyzed

B. Isolated Cell Staining Method

1. Isolate leukocytes from heparinized blood or tissue samples by Ficoll-Hypaque density gradient centrifugation.
2. Wash the cells twice with McCoy's and centrifuge at 300 x g for 5 minutes.
3. Resuspend the cells in McCoy's and adjust the concentration to 1 xl Oe6 cell/ml.
4. Incubate 100ui (lOe5 cells) with lOul of H6098-61A in the dark at 4°C for 20 minutes.
5. Wash the cells twice with 2ml PBS at 300 x g for 5 minutes.
6. For FITC conjugated Mab, go to step #8.
7. Repeat step 5.
8. Resuspend the pellet in O.5ml of fixing solution. The cells are ready for immediate flow cytometry analysis, or can be stored in the dark at 4°C for 24 hours before being analyzed.

Expeded Values
Each laboratory should establish the normal ranges of positive cells and negative cells under its own testing conditions for each batch of samples. FITC conjugated monoclonal antiboaies should show a FL 1 channel shift where it reacts with tfle positive cells when compared to the negative cells. Biotin conjugated monoclonal antibodies combined with the usage of streptavidin-PE should show a FL2 channel shift wnen reacting with the positive cells. Different FITC or Biotin conjugated monoclonal antibodies may have different ranges of channel shift for the positive reactions. In addition, individual lots of FITC or Biotin conjugated monoclonal antibodies may have different channel shifts for the positive reactions.

Specific Performance Charaderistics
Normal lymphocytes should all express HLA-Class I antigens. Therefore, lymphocytes should be selected by proper gates on FSC versus SSC dot plots for the studies of HLA-Class I antigens. B cells express both Class I and Class II antigen. T cells do not express class II antigens. Therefore, when Class II antigens are studied using the biotinylated monoclonal antibodies, selection of B cells using CD20-FITC with streptavidin-PE is the preferred method.

Small volumes of anti-HLA-C antibody vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

MBS603035 is an antibody targeting HLA-C. Also known as HLA Class 1 Antigen Bw4 (FITC). Reactive in Human. Host: Mouse.