Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma.
Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (>0.1 microm). We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size.
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. a.e.michelsen@biokjemi.uio.no
Annika E
AE
Michelsen
Ragnhild
R
Wergeland
Oddvar
O
Stokke
Frank
F
Brosstad
D000911:Antibodies, Monoclonal / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D001792:Blood Platelets / Q000737:chemistry*; D000001:Calcimycin / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005063:Europium / Q000378:metabolism; D005434:Flow Cytometry; D015200:Fluoroimmunoassay; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007554:Isotopes; D010316:Particle Size; D010446:Peptide Fragments / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D040201:Platelet Membrane Glycoprotein IIb / Q000032:analysis*; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism; D001620:beta-Thromboglobulin / Q000032:analysis
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D003902:Detergents; D007476:Ionophores; D007554:Isotopes; D010446:Peptide Fragments; D040201:Platelet Membrane Glycoprotein IIb; D001620:beta-Thromboglobulin; C082835:thrombin receptor peptide (42-47); D000001:Calcimycin; D005063:Europium; D001710:Biotin; D019809:Streptavidin