PROPERTIES OF MONOCLONAL ANTIBODIES TO HUMAN FACTOR IX-PHAGE CAP-SID FUSION PROTEIN
XIth International Congress on Thrombosis and Haemostasis
Functional domains of the human factor IX (FIX) molecule may be expressed in E. coli as fusion proteins (FPs) with phage T7 capsid protein 10 (CP 10). Such proteins are synthesized in large amounts and, because of their insolubility can be easily purified Polyclonal antibodies raised to FIX FPs cross react with the native molecule, but the utility of FPs in the production of monoclonal probes of native FIX is unclear. Hence, we have characterized the specificity of murine hybridomas resulting
... ridomas resulting from the immunization with a FP containing amino acids -3 to 49 of human FIX (FP4D) . FP4D proved to be a potent immunogen in mice. Using solid phase ELISA assays, 46% of 2,200 cultures screened reacted with the immunizing antigen. Most of the positive cultures reacted only with FP4D and/or CP 10 and were discarded. Fifty cultures reacted with native FIX and were further studied. None of the FIX reactive cultures inhibited FIX:C activity in clotting assays. Binding of more than half of the cultures to FIX was inhibited by the presence of 5mM Ca++ suggesting a similarity between FP4D and the non Ca++ bound steric state of FIX. Four cultures were subcloned 2X to insure monoclonality. These antibodies were further evaluated for binding to FIX, FP4D, and CP 10 in the presence or absence of Ca++. Three of the four Mabs reacted with FP4D and FIX and showed varying degrees of inhibition by Ca++ only with FIX. One antibody reacted equally with all three proteins in the presence or absence of Ca++. Two of the Mabs were detectable in a solid phase RIA and showed increased radioligand binding in the absence of Ca++. Most Mabs against FP4D appear to detect epitopes which are influenced by the presence of Ca++. Since the FIX amino acid sequence contained in FP4D is gammacarboxylated and capable of binding Ca++ in the native molecule, the epitopes detected by these Mabs may be masked or altered by Ca++ binding. These Mabs may prove to be useful probes in the study of Ca++ binding in the native FIX molecule.