EVALUATION OF IGG MOLECULES, FAB' FRAGMENTS AND IGG-HORSERADISH PEROXIDASE CONJUGATES AS SURFACE LABELS FOR FREEZE-ETCHED MEMBRANES

Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti‐SRBC IgG, antigen‐binding fragments of IgG (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labelled ghosts were deep‐etched and replicated with platinum‐carbon to visualize their surface features in the transmission electron microscope. The surfaces of control ghosts contain a very low number of ‘background’ particles (42 ± 8 particles/μm2) that vary in size from 4·5 to 25 nm. After labelling with whole IgG the density of surface particles (average diameter 12–3 nm) increases dramatically to 480 ± 54 per μm2. Fab'‐labelled ghosts exhibited both significantly fewer (87 ± 14 particles/μm2) and smaller (average diameter 9·8 nm) surface particles. Ghosts labelled with IgG‐HRPO conjugates possessed 590 ± 45 particles/μm2 with an average diameter of 15·3 nm. When these ghosts were incubated with diaminobenzidine and hydrogen peroxide the average size but not the density of the particles increased. Based on these and other observations we conclude that an organic surface marker for freeze‐etched membranes has to have a diameter of > 15 nm if it is to be consistently seen over extended areas and against the background granularity of the surface of a red blood cell ghost. Somewhat better resolution may be expected if markers consisting of inorganic crystals with a distinct shape and coupled to Fab' fragments can be made. 1979 Blackwell Science Ltd