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A fluorescence-conjugated immunobinding assay for the detection of P-selectin on platelets
D Wen1, T T Nguyen, E A Plumhoff
1Department of Endocrinology, Mayo Clinic, Rochester, Minnesota.
Insights
A new fluorescence-conjugated immunobinding assay (FCIBA) accurately measures P-selectin on activated platelets. This rapid assay shows excellent precision and correlates well with flow cytometry, offering a convenient method for evaluating platelet activation.
Area of Science:
- Hematology
- Immunology
- Biochemistry
Background:
- P-selectin, also known as GMP-140 or PADGEM, is a protein translocated to the platelet plasma membrane upon activation.
- P-selectin expression serves as a potential biomarker for assessing platelet activation status.
Purpose of the Study:
- To develop and validate a fluorescence-conjugated immunobinding assay (FCIBA) for the specific detection of P-selectin on human platelets.
- To evaluate the precision, speed, and correlation of the FCIBA with established methods like flow cytometry.
Main Methods:
- Human platelets were isolated and stimulated with varying doses of adenosine diphosphate (ADP).
- Fixed platelets were incubated with a fluorescence-conjugated anti-P-selectin monoclonal antibody.
- Fluorescence intensity was measured using a fluorescence concentration analyzer.
Main Results:
- The FCIBA demonstrated high precision with intra-assay and inter-assay coefficients of variation below 8.11%.
- The assay showed excellent correlation (r = 0.936) with flow cytometry for P-selectin measurement.
- P-selectin translocation was dose-dependent on ADP stimulation and correlated with platelet aggregation.
Conclusions:
- The developed FCIBA is a fast, precise, and convenient method for determining P-selectin expression on human platelets.
- This assay provides a reliable tool for evaluating platelet activation, with results comparable to flow cytometry.
Abstract:
P-selectin (GMP-140 or PADGEM) is translocated to the plasma membrane of platelets after platelet activation. P-selectin, therefore, may be a potential marker for evaluating platelet activation. A fluorescence-conjugated immunobinding assay (FCIBA) has been developed to detect specifically P-selectin on platelets. Platelets were isolated from fresh blood by centrifugation and stimulated with various doses of ADP before being fixed with 1% of paraformaldehyde. Fixed platelets were incubated with fluorescence-conjugated anti-P-selectin monoclonal antibody in the wells of fluoricon microtiter plates, and the fluorescence intensity was read on a fluorescence concentration analyzer. Once platelets were fixed, the procedures were completed in < 2 hours. The intra-assay coefficient of variation (CV) was 6.97% (n = 40), the time-based interassay CV was 8.11% (n = 16), and the sample-based inter-assay CV was 6.17% (n = 16). The FCIBA had an excellent correlation (r = 0.936, p < 0.001) with flow cytometry in the measurement of expressed P-selectin in platelets of 20 normal donors. Translocation of P-selectin in plasma-suspended platelets in response to increasing doses of adenosine diphosphate (ADP) occurred in a dose-dependent manner and correlated positively with ADP-induced platelet aggregation in terms of both stimulating doses of ADP (r = 0.99, p < 0.01) and time intervals (r = 0.92, p < 0.05). The findings show that FCIBA is a fast and convenient assay with good precision for the determination of P-selectin expression of human platelets.
