Direct fluorescence-conjugated immunoassay for platelet activation

A fluorometric method for measuring platelet activation in a sample of platelets which is not exogenously activated comprising:

• (a) isolating in vitro a first sample of physiological material comprising platelets and a second sample of physiological material comprising platelets from a mammal, wherein said sample each contains a preselected number of platelets;
• (b) adding an amount of an activation agonist to said first sample in a liquid medium for a period of time effective to maximally activate the activatable platelets in said first sample;
• while maintaining the second sample in a liquid medium for an equivalent period of time;
• (c) forming binary labelled complexes with the activated platelets in each sample by adding to each sample an amount of
• (i) an anti-P-selectin antibody conjugated to a fluorescent label;
• or
• (ii) an anti-P-selectin antibody conjugated to a binding site for a detectable label followed by a detectable fluorescent label which specifically binds to said binding site;
• and
• (d) determining the fluorescence of the binary labelled complexes in each sample, wherein a ratio of the fluorescence of said second sample to said first sample provides a measure of the extent of platelet activation in said second sample.