FITC-conjugated anti-human fibrinogen antibodies.
Cat. # 2871

FITC-conjugated anti-human fibrinogen antibodies are intended to be used for flow cytometric measurement of platelet activation, spontaneous or induced, utilising fibrinogen binding as marker of platelet activation.

Price: 230 Euro

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Reagent for up to 100 test

For Research Use Only

Intended Use
FITC-conjugated anti-human fibrinogen antibodies are intended to be used for quantification of platelet activation, as measured by fibrinogen binding using flow cytometry.

1.0 Background

Platelets play a central role in the haemostatic system, and platelet aggregation has been linked to thrombotic diseases [1-3]. Platelet function is dependent upon membrane glycoprotein receptors and their interactions with plasma proteins and adhesive proteins on the vessel wall. Platelet activation causes a structural change in the GPIIb/IIIa complex that exposes the fibrinogen binding site, which subsequently binds fibrinogen. This binding is essential for platelet aggregation.

Platelet function in severely thrombocytopenic patients can not be determined using traditional techniques such as template bleeding time or aggregometry. Binding of fibrinogen to platelets can however be measured as an activation marker utilising flow cytometry [4]. By using FITC-conjugated chicken antibodies the problem with interference from complement components can be avoided. As low as 1 % activated platelets can be detected without interference from Fc‑interaction.

1.1 Clinical Indications

The measurement of fibrinogen binding to platelets has been shown to be of clinical relevance in patients with cardiovascular disease, e.g. myocardial infarction and angina pectoris [3]. Spontaneous activation of platelets can occur in diseases such as diabetes [5, 6], myeoloproliferative disorders[7] and sepsis [8]. The method may be used to monitor treatment with ADP-receptor antagonists [9]. Porcine platelets may also be studied [10].

Platelet transfusions are often used in the treatment of trombocytopenic patients, i.e. those undergoing treatment with cytostatics.

1.2 Assay Principle

Platelet rich plasma (PRP) from patients or healthy control subjects is incubated with buffer and FITC-conjugated anti-human fibrinogen. The FITC-anti-fibrinogen antibody will bind to activated platelets by binding to exposed GPIIb/IIIa receptors through the receptor ligand, fibrinogen. The amount of fluorescence bound to the platelets is used to determine the proportion of activated platelets in the PRP. As negative control to distinguish between activated and non-activated platelets, platelets incubated with buffer containing EDTA and FITC-anti-fibrinogen is used, since EDTA prevents the binding of fibrinogen to GPIIb/IIIa. The degree of activation is determined both for non-stimulated platelets and platelets stimulated with different concentrations of ADP or other agonists.

2.Reagents Provided

FITC-conjugated anti-fibrinogen: 1 vial of 1 mL ready to use affinity purified chicken- anti-fibrinogen 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 conjugated antibody should be stored dark at +2-6°C.

3.Warning

The FITC-conjugated antibody solution contains 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.

4.Reagents and Equipment required but not Provided

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.
HEPES/EDTA-buffer: HEPES-buffer with 10 mmol/L EDTA.
ADP: 1.7 and 8.5 µmol/L in HEPES-buffer
Plastic tubes, 2.5 / 5 mL capacity
Flow cytometer

5.Sample Collection

Nine volumes of venous blood are collected in 1 volume of 0.1 M trisodium citrate. Prepare platelet rich plasma (PRP) by centrifugation at 140 x g for 10 minutes at room temperature. For determination of spontaneous platelet activation, the sample must be assayed immediately. For determination of activation after stimulation with ADP, the samples should be kept at room temperature and analysed between 0.5 and 4 h after sampling.

6.Assay Procedure

6.1 Preparation of Negative Control

Add to a plastic tube:
230 µL HEPES/EDTA-buffer
10 µL FITC-conjugated anti-fibrinogen
20 µL PRP

Incubate for 10 minutes at room temperature (20-25 °C)
Add 20 µL HEPES/EDTA-buffer
Incubate for exactly 10 minutes at room temperature (RT)
Add 2 mL ice cold HEPES-buffer
Analyse on a flow cytometer within 6 h.

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

6.2 Preparation of Samples

Dissolve ADP in HEPES-buffer to final concentrations of 0, 1.7 and 8.5 µM respectively. For each sample, 20 µL of the respective solutions is needed.

Add to three plastic tubes:
230 µL HEPES-buffer
10 µL FITC-conjugated anti-fibrinogen
20 µL PRP

Incubate for 10 minutes at RT
Add 20 µL of the respective ADP solutions (0, 1.7 and 8.5 µM) to the three tubes.
Incubate for exactly 10 minutes at RT
Add 2 mL ice cold HEPES-buffer
Analyse on a flow cytometer according to the instructions of the manufacturer. The analytical markers in the fluorescence channel are used to divide the negative control sample into two fractions containing 98-99 % of the platelets and 1-2 % of the brightest representatives of the platelet population. Platelets with fluorescence greater than the marker are identified as positive events.

The fraction (in %) activated platelets at different ADP concentrations are calculated and compared to the degree of activation with and without stimulation for healthy normal subjects.

7.Limitations

Vigorous stirring of platelets must be avoided. At low concentrations of fibrinogen (below 0.3 g/L) immune complexes are formed which may result in increased binding of fibrinogen and antibody to the platelets [4]. When estimating fibrinogen binding to platelets in patient samples suspected to posses low concentrations of fibrinogen, additional safety could be achieved by determining the fibrinogen concentration.

8.Quality Control

The amount of label has been quantified by comparison with FITC-labelled standards.
9.References
1. Vreeken, J., et al.: Spontaneous aggregation of blood platelets as a casue of idiopathic thrombosis and recurrent painful toes and fingers. Lancet, i:1395-1397, 1971.
2. Wu, K., et al.: Spontaneous platelet aggregation in arterial insufficiency: Mechanisms and implication. Thromb. Haemostas., 35:702-711, 1976.
3. Yi: Blood Coagulation and Fibrinolysis,5:64, 1994.
4. Lindahl, T., et al.: Studies of fibrinogen binding to platelets by flow cytometry: An improved method for studies of platelet activation. Thromb. Haemostas., 68:221-225, 1992.
5. Tschoepe, D., et al.: Evidence for abnormal platelet glycoprotein expression in diabetes mellitus. Eur. J. Clin. Invest., 20:166-170, 1990.
6. Tschoepe, D., et al.: Large platelet circulate in an activated state in diabetes mellitus. Sem. Thromb. Hem., 17:433, 1991.
7. Wehmeier, A., et al.: Circulating activated platelets in myeoloproliferative disorders. Thromb. Res., 61:271-278, 1991.
8. Lundahl T., et al:: Blood Coagulation and Fibrinolysys 7:218-20,1996.
Berglund U, Lindahl T. Enhanced onset of platelet inhibition with a loading dose of ticlopedine in ASA-treated stable coronary patients. Int J Cardiology 64:215-17,1998
Larsson A, Eriksson M, Lindahl TL. Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet cativation. Platelets ., 13: 153-157,2002.

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