Purification of PDGF-AB and PDGF-BB from human platelet extracts and identification of all three PDGF dimers in human platelets.
We have developed a panel of monoclonal antibodies to platelet-derived growth factor (PDGF) which have variable specificities for the three dimeric forms of the molecule (AA, AB, and BB). We have used these antibodies to detect and immunoaffinity purify the individual dimers from human platelet rich plasma. Extracts of outdated platelet preparations were initially chromatographed over CM-Sepharose and then passed over the Sepharose-coupled monoclonal antibodies in series in selectively isolate the three dimeric forms of PDGF. The PDGF eluted from the affinity columns was subsequently further purified by reversed-phase HPLC. From 300 units of outdated platelet preparations, we purified 58 micrograms of PDGF-BB and 140 micrograms of PDGF-AB. Using the monoclonal antibodies to develop PDGF dimer-specific ELISAs, it was observed that all three PDGF dimer forms are present in fresh human platelet extracts and that the ratios of the three dimer forms vary depending upon the extraction conditions used. The identification of all three PDGF dimer forms in human platelets point to the need to view PDGF isolated from human platelets by conventional techniques as a mixture of all three forms and not solely as PDGF-AB.
Hart
C E
CE
ZymoGenetics Inc., Seattle, Washington 98105.
Bailey
M
M
Curtis
D A
DA
Osborn
S
S
Raines
E
E
Ross
R
R
Forstrom
J W
JW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D001792:Blood Platelets / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008934:Mitogens / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000145:classification*
D000911:Antibodies, Monoclonal; D008934:Mitogens; D010982:Platelet-Derived Growth Factor