FITC-Conjugated anti-human platelets chicken antibodies.
Cat. # 2872

FITC-conjugated anti-human platelet antibodies are intended for quantification of platelets by flow cytometry.

Price: 220 Euro

Order now at mail: Info@diapensia.se

Reagent for up to 100 test

For Research Use Only.

Intended Use
FITC-conjugated anti-human platelet antibodies are intended for quantification of platelets by flow cytometry.

1.Background and Clinical Indications

Platelets play a central role in the hemostatic system. In many cases of coagulation disorders, it is of interest to quantify the number of platelets. The measurement of total platelet numbers is of clinical relevance in patients with e.g. thrombocytopenia. Due to the immunological differences between birds and mammals, chicken antibodies do not initiate complement activation in humans. Furthermore, the large phylogenetic difference between mammal and avian species also ensures a high antibody titer [1].

2.Assay Principle

Whole blood samples are incubated with buffer and FITC-conjugated anti-platelet antibodies, allowing the binding of the antibodies to platelets. The platelet concentration is then determined using fluorescence activated flow cytometry. As negative control, FITC-conjugated chicken IgG (anti human insulin) is recommended (Cat # 2873).

3.Reagents Provided

FITC-conjugated anti-platelet antibodies: 1 vial of 1 mL ready to use affinity purified chicken anti-platelet antibodies, conjugated with fluorescein isothiocyanate (FITC). The solvent is 0.02 M sodium phosphate, 0.15 M NaCl and 0.02 % sodium azide, pH 7.2. The FITC/Protein (F/P) ratio is 3.5 ± 1.0. The antibody should be stored in the dark at +2‑6°C.

4.Warning

The FITC-conjugated antibody solution contain sodium azide which may react with lead and copper plumbing to form highly explosive metal azides. Materials discarded into the sink should be flushed with a large volume of water to prevent azide build-up.

5.Reagents and Equipment Required but not Provided

Lysing solution, Diapensia cat. # 2970
Control antibody: FITC conjugated chicken anti-human insulin, Diapensia cat. # 2873
HEPES-buffer: 20 mmol/L HEPES, 137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 5.6 mmol/L glucose, 1 g/L bovine serum albumin, pH 7.40. Plastic tubes, 2.5 / 5 mL capacity
Flow cytometer


6.Sample Collection

Nine volumes of venous blood are collected in 1 volume of 0.1 M trisodium citrate. EDTA samples may also be used. The samples should be analysed between 0.5 and 4 h after sampling.

7.Procedure

7.1 Preparation of Negative Control

Add to a plastic tube:
100 µL HEPES-buffer
10 µL FITC-conjugated chicken IgG
10 µL whole blood

Incubate for 10 minutes at room temperature (20-25 °C).

Add 10 µL HEPES-buffer.

Incubate for exactly 10 minutes at room temperature (RT).

Add 1 mL of lysing solution (Biopool cat # 2970).

Incubate for 10 minutes.

Dilute sample 20x with lysing solution, i.e. 100 µL sample with 1900 µL lysing solution.

Samples should be analysed on a flow cytometer within 4 h.

Note!

In some flow cytometers the sample should be further diluted in a buffer of the manufacturers choice.

7.2 Preparation of Samples

Add to plastic tubes:
100 µL HEPES-buffer
10 µL FITC-conjugated anti-platelet antibodies
10 µL whole blood

Incubate for 10 minutes at RT.

Add 10 µL HEPES-buffer.

Incubate for exactly 10 minutes at RT.

Add 1 mL of lysing solution (Diapensia Cat # 2970).

Incubate for 10 minutes.

Dilute sample 20x with lysing solution, i.e. 100 µL sample with 1900 µL lysing solution.

Analyse on a flow cytometer within 4 h.

8.Quality Control

The amount of label has been quantified by comparison with FITC-labelled standards.

9.Correlation to Microscopical Methods

Flow cytometry counting was compared to microscopy [2] and found to correlate well (r = 0.94). The CV was also lower with flow cytometry.

10.References

1. Larsson, A., et al.: Taking advantage of evolution - A review. Poult. Sci., 72:1807-1812, 1993.
2. Lindahl, T., et al.: A new method for measurement of platelet concentration using flow cytrometry, in XXIV Nordic congress in Clinical Chemistry. 1994: Stockholm, Sweden.

Ver 030617