Electron microscopic studies of the antigen-antibody complex.

Electron micrographs of the ferritin antibody (rabbit) and ferritin (horse) complex have been obtained. The high iron content of the ferritin molecule (23 per cent Fe) allows its molecules to be recognized within the particles of precipitate. Three methods of visualizing the molecular distribution have been developed: () small particles of the precipitated complex have been dried on to electron microscope grids and either examined directly or first shadowed with metal and then examined, () the precipitate has been centrifuged to a plug which was embedded and thin sections cut from it for examination, () the bands formed by allowing antibody and antigen to diffuse together in agar gels have been fixed, embedded and sectioned. All methods have yielded pictures of the distribution of the ferritin within the complex which are broadly similar to what might have been expected from a somewhat irregular lattice as pictured in the Marrack-Heidelberger Lattice Theory. The antibody molecules are not clearly defined but appear as a halo of low density enveloping the ferritin clusters. The distance, centre to centre, between the ferritin molecules is variable, but is, on the average, in the range 200–400 Å. This is greater than the ferritin-ferritin contact distance (100 Å) and is thought to mean that the ferritin molecules are bridged by antibody molecules as pictured in the Lattice Theory. The bands produced in the gel-diffusion test contain islands of ferritin-antibody complex. When equivalent concentrations of reagents are used a single band of precipitate is formed. When excess of either antigen or antibody is used multiple bands of precipitate are formed which contain islands of ferritin antibody complex indistinguishable from those formed in the single band at equivalent concentrations, providing direct evidence for the formation of multiple bands from a single antigen. Ferritin-ferritin contacts have been observed within the complex. Under all the conditions of relative concentration of the two components used here, the particles of precipitated complex seem to be superficially covered with antibody which is seen as a halo about 300–400 Å thick around each cluster. This distance may correspond to the length of the antibody molecule which is deduced from other measurements to be about 300 Å.