rev: March 13, 1998

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  ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, 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.


ANTI-Human Fetal Hemoglobin
DATA SHEET: Mouse anti-Human Fetal Hemoglobin

Catalog#: RDI-FHBabm         $375.00/100ug

                RDI-FHBabmFT    $438.00/100tests

                RDI-FHBabmFTX  $1250.00/400T

Package Size: 100ug IG1 in 0.5ml PBS w/0.1%NaN3

-conjugated material provided in BSA at approx 4-5mg/ml


Clone: B1


Species: mouse IgG1

Reactivity: reacts with human fetal hemoglobin in flow cytometry (the main cellular reactivities are fetal red cells).


Uses: -flow cytometry (use approx 10ul to label 100K RBC's )Titer must be optimized for each method


Storage: Before use, tighten cap, centrifuge vial for 1-2 minutes at 1000 rpm to concentrate antibody in vial. Store at 2-8 DEG C for up to 3 months, or aliquot and store at -10 DEG C or colder for long term. Avoid frequent freeze thaw cycles. Protect Conjugated antibodies from light.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics. Sodium azide may form explosive compounds in presence of heavy metals or under acidic conditions. Flush drains with copious amounts of water to prevent buildup of explosive compounds. 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.


Quantitation of erythrocytes containing fetal hemoglobin (HbF) is used to determine the extent of a fetal-to-maternal hemorrhage. This is important in estimating dosage of RhIg for RhD-negative mothers, but also in evaluating complicated pregnancies or those involving trauma, and explaining fetal or newborn distress.

-This antibody is strictly available for research project use only-


Sample Protocol Using Fitc conjugated Antibody:

Specimen:

Well mixed EDTA anticoagulated whole blood

Refrigerate specimen if testing is not performed within 4 hours  post collection.

Specimens may be held refrigerated for 5 days prior to testing.


Materials & Equipment:

1. 12x75 mm disposable polystyrene tubes (Falcon #2052) with rack.

2. Pipette tips, yellow (1-200 ul) and blue (200-1000 ul).

3. Various adjustable pipettes (5-40, 40-200, and 200-1000 ul).

4. Vortex mixer.

5. DAC II cell washer (Baxter)  Or Equivalent  (Manual Washing May also be used)

Note: Flow Cytometric sheath fluid is the reagent used with the cell washer. In the absence of a cell washer, wash cells with 2ml of PBS-0.1%BSA and centrifuge at 1500 RPM for 5 minutes.

6. Multipurpose flow cytometer


Procedure:

VORTEX: In this procedure, when instructed to vortex, do so at high speed for 15 seconds.

1. Measure the RBC count of the specimen .

2. Fix X ul of whole blood in 1 ml of cold 0.05% Glutaraldehyde for 10 minutes at room temperature. Vortex after the addition of cells.

X is approximately 2.5 x10 E07 RBCs. This number of RBCs in the fix tube will result in approximately 500K cells in the stain tube. If the RBC count is > 5.0x106/ul, dilute 1:2 with PBS-0.1%BSA and remeasure the RBC count. Determine X.

3. Load the tubes into the automatic cell washer and select the three wash cycles. Start the cell washer.

4. Resuspend the cell pellet by vortex in 0.5 ml 0.1% Triton X100. Incubate for 3-5 minutes at room temperature.

5. Load the tubes into the automatic cell washer and select theone wash cycle. Start the cell washer.

6. Resuspend the cell pellet in 0.5 ml PBS-BSA by vortexing or gentle pipettiing

7. Add 10 ul of this suspension (about 1-2x105 cells) to 20 ul**of the working antibody dilution and 70ul of PBS-0.1%BSA.  Incubate at room temperature in the dark for 15 minutes.

8. Load the tubes into the automatic cell washer and select the two wash cycle. Start the cell washer.

9. Resuspend cell pellet by vortex in 0.5 ml of 1% Formaldehyde.Store tubes in the dark in the refrigerator until ready for flow cytometric acquisition.

Reagents:

1. PBS - 0.1%BSA

Phosphate buffered saline (PBS, Sigma #1000-3)

qs packet to 1 L with deionized H20

add 1 g of Bovine Albumin (BSA, Sigma #3294)

pH to 7.4

Store in refrigerator

Expiration: 7 days

2. 0.05% Glutaraldehyde

Dilute 25% Glutaraldehyde (Sigma #g5882) with PBS to .05%

Glutaraldehyde

100ul diluted to 50 ml

Store stock (25% Glutaraldehyde) in freezer

Expiration: See container

Store working solution (0.05% Glutaraldehyde) in refrigerator

Expiration: make fresh each day of use

Use cold

3. 0.1% Triton X-100

Dilute Triton X-100 (Sigma #X100) to 0.1% with PBS-0.1%BSA 50ul diluted to 50ml

Store stock (Triton X-100) at room temperature

Expiration: See container

Store working solution (0.1% Triton X-100) in refrigerator

Expiration: 30 days or visible growth Use cold

4. 1% Formaldehyde

Dilute 10% Formaldehyde - Methanol Free(Polysciences, Inc. #04018) with PBS-0.1%BSA to 1% pH to 7.4

Store stock (10% Formaldehyde - Methanol Free) at room  temperature

Expiration: See container

Store working solution (1% Formaldehyde) in refrigerator

Expiration: 7 days Use cold

5. Alsevers's Solution

0.33 g Chloramohenicol

0.50 g Citric Acid C6H8O7

20.5 g Dextrose C6H12O6

2.0 g Inosine

0.5 g Neomycin sulphate

4.2 g Sodium chloride NaCl

8.0 g Trisodium citrate C6H5Na3O7.2H2O

1. Dissolve citric acid, detrose, sodium chloride and trisodium citrate in approximately 600 mL of distilled water.

2. Add chloramphenical, inosine and neomycin sulphate; mix well.

3. Dilute to 1 L with deionized water.

Store in refrigerator

Expiration: 90 days or visible growth

6. Anti-Hemoglobin-F antibody FITC conjugated  (cat#RDI-FHBabmFT)

Dilute current lot # 1+1 with PBS-0.1%BSA (Optimiuze for each batch) Dilute only the amount to be used for one day

Protect from light

Store stock antibody in the refrigerator protected from light

Expiration: one year

References:

1. Sebring ES, Polesky HF. Fetomaternal Hemorrhage: Incidence, Risk Factors, Time of Occurence, an Clinical Effects. Transfusion. 1990, 30:344-357.

2. Davis BH. Flow Cytometric Analysis of Red Blood Cells. Clinical Flow Cytometry: Principles and Application, pp.373-387.  Williams & Wilkins, 1993.

3. Nance SJ, Nelson JM, Arndt PA, et.al. Quantitation of Fetal-Maternal Hemorrhage by Flow Cytometry. American Journal of Clinical Pathology. 1989, 91: 288-292.

4. Medearis AL, Hensleigh PA, Parks DR, Herzenberg LA.  Detection of Fetal Erythrocytes in Maternal Blood Post Partum with the Fluorescence-Activated Cell Sorter. American Journal of Obstetrics and Gynecology. 1984, 148:290-295.

5. Thorpe SJ, Thein SL, Sampietro M, et.al. Immunochemical Estimation of Hemoglobin Types in Red Blood Cells by FACS Analysis. British Journal of Hematology. 1994, 87:125-132.

6. Bauer KD, Jacobberger JW. Analysis of Intracellular Proteins. Methods in Cell Biology. 1994, 41:351-376.

7. Clevenger CV, Shankey TV. Cytochemistry II: Immunofluorescent Measurement of Intracellular Antigens. Clinical Flow Cytometry: Principles and Application, pp.157-177.  Williams & Wilkins, 1993.

8. Freedman J, Lazarus AH. Applications of Flow Cytometry in Transfusion Medicine. Transfusion Medicine Reviews. 1995, 9:87-109.

9. Pattanapanyasat K, Udomsangpetch R, Webster HK. Two-Color Flow Cytometric Analysis of Intraerythrocytic Malaria Parasite DNA and Surface Membrane Associated Antigen in Erythrocytes Infected with Plasmodium falciparum. Cytometry. 1993, 14:449-454.

10. Olsen SH, Bigelow NC, Davis BH, Chen JC, Bagwell,CB. Flow Cytometric detection of fetal hemoglobin (HbF) - containing red blood cells. Laboratory Hematology. 1996,1:74

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RDI Divison of Fitzgerald Industries Intl

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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