| Many attempts to raise monoclonal antibody (MoAb) against human Platelet Factor 4 (PF4) were unsuccessful due to the difficulty of obtaining MoAbs to such a low molecular weight and highly conserved protein as PF4. In our laboratory, BabL/c mice were immunized first in intraperitoneal routes and boosted with intrasplenic immunization to stimulate antibody response to minute amounts of PF4. After hybridizing and screening, we elicited a novel MoAb against human PF4 having stable and high titers. In Indirect ELISA and Dot blot, this MoAb, SZ-95, which is characterized as IgG1, only recognized human PF4 and did not react with other CXC chemokines such as beta-thromboglobulin ( P -TG) and the synthetic peptide corresponding to amino acids 34 to 58 (P34-58). Further study in Western blot in reducing condition, showed that the change of tertiary structure of nature PF4 did not affect SZ-95 binding to PF4. Therefore, human PF4 can be purified from triton X-lOO solubilized platelet solution using SZ-95-sepharose 4B affinity chromatography prepared by us. Besides, we obtained rabbit anti-PF4 polyclonal antibodies with purified PF4 and study the heparin binding properties of PF4 using SZ-95 and anti- PF4 polyclonal antibodies at the same time. Our heparin:PF4 ELISA demonstrated that the quantities of both SZ-95 and anti-PF4 polyclonal antibodies reacting with heparin:PF4 complexes is smaller those with PF4. Moreover, the more heparin in complexes, the less quantities it showed. This result not only indicated the epitope, which SZ-95 recognized, may be important in heparin binding to PF4, but also the II Preparation and Application of a Monaclonal Antibody against Human Platelet Factor 4 ABSTRACT change of function and structure of PF4 after binding maybe depend on the dose of heparin. This novel MoAb specifically reacting with hPF4 and also seems capable of interfering with PF4抯 biological activities, provides a powerful means to understand the structure/function relationship of this protein.
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