IMMUNOASSAY OF HUMAN PLATELET FACTOR-4 (PF4, ANTIHEPARIN FACTOR) BY RADIAL IMMUNODIFFUSION

Abstract

PF4 antigen was measured in platelets and in plasma by means of single radial immunodiffusion. Anti-PF4 antibody obtained in rabbits by injecting highly purified human PF4 was monospecific in double immunodiffusion and in quantitative rocket immunoelectrophoresis. A high degree of correlation was observed between the precipitation zones in the radial immunodiffusion method and the amount of purified PF4 (in the range of 0.6-50.0 .mu.g/ml) or the number of platelets in plasma (in the range of 5 .times. 106-1.6 .times. 108 plateiets/ml applied. The sensitivity of the method was 30-125 times higher as compared with clotting assay (antiheparin activity), and the standard error of the method was 2.3%. The method was specific for the antigen present in platelets since human leukocytes and erythrocytes gave negative results. Release of PF4 antigen from washed platelets challenged with thrombin, collagen, ADP and antigen-antibody [rabbit] complexes was measured by the radial immunodiffusion assay. It usually paralleled the release of 3H-serotonin, but PF4 antigen was a more sensitive marker for platelet release reaction. Release of PF4 antigen was usually 2-4 times higher than release of the antiheparin activity as measured by clotting assay when both were compared as percentage of total content in platelets. The level of PF4 antigen was determined in platelet-rich plasma (PRP) and platelet-free plasma (PFP) obtained from 12 healthy volunteers. While the mean level of extraplatelet pool of PF4 antigen in PFP was 0.72 .+-. 0.92 .mu.g/ml, PRP contained 80 .+-. 22 .mu.g of PF4 antigen/109 platelets. Addition of thrombin (1 U/ml) liberated all of the PF4 antigen (78 .+-. 24 .mu.g) present in PRP, but ADP (50 .mu.M) released only 31 .+-. 22 .mu.g of PF4 antigen/109 platelets. The presence of heparin did not interfere with the assay of intraplatelet or extraplatelet PF4 by single radial immunodiffusion. The method described represents a simple, sensitive, quantitative and specific assay for human PF4 antigen possessing antiheparin activity.