Purification of antibodies labelled with monomeric ferritin, and use in the localisation of Salmonella flagellar antigens

Introduction: In immuno-electron microscopy, electron-dense labels are required to permit localisation of the bound antibody molecules. One of the most successful is ferritin [1-3]. If conjugated antibody is present in the immunolabelling mixture, it must have the effect of diminishing sensitivity, perhaps critically, by competing for the antigenic sites. Therefore, it is important to remove free antibody from the conjugate preparation. Unconjugated ferritin may also interfere by giving a high background of non-specific 'staining' , so that this should be removed. Conventional preparations of ferritin, e.g. from horse spleen, are heterogenous in that some of the spherical units exit as dimers, trimers and higher oligomers [4-6]. In addition, isoelectric focusing has demonstrated a further type of heterogeneity in ferritin; multiple band patterns being observed in ferritins from single organs f7, 8]. This latter phenomenon is not significant for the present work. The presence of oligomers in the conjugate preparation may give rise to errors in interpretation because of a false impression of close grouping of bound antibody molecules. In summary, therefore, only monomer should be used for conjugation and the conjugated product should be rigorously separated both from free ferritin and from free antibody. Attempts at purification of ferritin-antibody conjugates by chromatography on DEAE-cellulose and on sephadex columns have not been entirely successful [2, 9]. Purification has also been attempted by curtain electrophoresis and by electrophoresis on agar [10, 11]. Neither method has been successful in fully resolving the crude mixture of monomeric and polymeric ferritin, ferritin antibody and uncoupled antibody globulin. Polyacrylamide gel electrophoresis, however, offers excellent separation of monomer ferritin from oligomers and we have earlier shown that preparative electrophoresis can give essentially pure monomer in a single step [5]. In the present report we describe the use of preparative electrophoresis for the purification of antibody-(monomeric) ferritin conjugate, from the conjugation mixture.