Niewiarowski S, Lowery C T, Hawiger J, Millman M, Timmons S
J Lab Clin Med. 1976 Apr;87(4):720-33.
Human platelet factor 4 antigen (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 to 50.0 mug per milliliter) or the number of platelets in plasma (in the range of 5 x 10(6) to 1.6 x 10(8) platelets per milliliter applied. The sensitivity of the method was 30 to 125 times higher as compared with clotting assay (antiheparin activity) and the standard error of the method was 2.3 per cent. 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 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 to 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 mug per milliliter, PRP contained 80 +/- 22 mug of PF4 antigen per 10(9) platelets. Addition of thrombin (1 U. per milliliter) liberated all of the PF4 antigen (78 +/- 24 mug) present in PRP but ADP (50 muM) released only 31 +/- 22 mug of PF4 antigen per 10(9) 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.
采用单向辐射免疫扩散法测定人血小板第4因子抗原（PF4抗原）在血小板及血浆中的含量。通过注射高度纯化的人PF4在兔体内获得的抗PF4抗体，在双向免疫扩散及定量“火箭”免疫电泳中均具有单特异性。在单向辐射免疫扩散法中观察到沉淀区与纯化PF4的量（每毫升0.6至50.0微克范围内）或血浆中血小板数量（每毫升加入5×10⁶至1.6×10⁸个血小板范围内）之间存在高度相关性。该方法的灵敏度比凝血测定法（抗肝素活性）高30至125倍，方法的标准误差为2.3%。该方法对血小板中存在的抗原具有特异性，因为人白细胞和红细胞检测结果为阴性。通过单向辐射免疫扩散测定法检测经凝血酶、胶原、ADP及抗原 - 抗体复合物刺激的洗涤血小板中PF4抗原的释放。其通常与³H - 5 - 羟色胺的释放平行，但PF4抗原是血小板释放反应更敏感的标志物。当将PF4抗原释放量与凝血测定法测得的抗肝素活性释放量作为血小板总含量的百分比进行比较时，PF4抗原的释放量通常比抗肝素活性释放量高2至4倍。测定了12名健康志愿者的富血小板血浆（PRP）和无血小板血浆（PFP）中PF4抗原的水平。PFP中PF4抗原的血小板外池平均水平为每毫升0.72±0.92微克，PRP中每10⁹个血小板含有80±22微克PF4抗原。加入凝血酶（每毫升1单位）可释放PRP中存在的所有PF4抗原（78±24微克），但ADP（50微摩尔）每10⁹个血小板仅释放31±22微克PF4抗原。肝素的存在不干扰单向辐射免疫扩散法对血小板内或血小板外PF4的测定。所描述的方法是一种针对具有抗肝素活性的人PF4抗原的简单、灵敏、定量且特异的测定方法。
