The development of a quantitative enzyme-linked immunosorbent assay to detect human platelet factor 4.
BACKGROUND
Platelet factor 4 (PF4) is a marker for in vitro and in vivo tests of platelet (PLT) activation and alpha-granule secretion. PF4 is also a major CXC cytokine released during storage. Cytokines released during PLT storage are a potential cause of nonhemolytic transfusion reactions. Quantitative measurement of PF4 requires an assay that is both reliable and sensitive. To achieve this goal, a sensitive, cost-effective, sandwich enzyme-linked immunosorbent assay (ELISA) was developed with commercially available antibodies to human PF4.


METHODS
An ELISA was developed for measuring PF4 from whole human PLTs or secreted from activated PLTs. Optimal concentrations of capture antibody, detection antibody, and enzyme-conjugate were determined with serial twofold dilutions of recombinant PF4. This assay was used to determine the ideal sample dilutions needed for reliable quantitation of PF4 in releasates or from whole PLT extracts.


RESULTS
Serial dilutions of recombinant PF4 resulted in a sigmoid titration curve with a maximal sensitivity of 10 pg and a dynamic quantitative range from 100 to 2500 pg. This ELISA was used to measure secretion from permeabilized PLTs stimulated with free calcium. In a secretion experiment with 2.5 x 10|*bsup*|8|*esup*| PLTs per mL, samples required a 1:10-fold dilution to reliably evaluate alpha-granule release.


CONCLUSIONS
The parameters described yield an ELISA method with low background and high sensitivity over a range of PF4 concentrations. Using the commercial reagents described makes this assay cost-effective and therefore suitable for analyzing multiple samples in the research setting.
Schraw
Todd
T
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
Whiteheart
Sidney
S
D000906:Antibodies; D001426:Bacterial Proteins; D015415:Biomarkers; D001792:Blood Platelets / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006735:Horseradish Peroxidase; D006801:Humans; D015539:Platelet Activation; D010978:Platelet Factor 4 / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D015415:Biomarkers; C058865:Strep-avidin conjugated horseradish peroxidase; D010978:Platelet Factor 4; D006735:Horseradish Peroxidase