A Two-Layer Technique for Detecting Surface Antigens in the Sea Urchin Egg With Ferritin-Conjugated Antibody

Budding of Frog Virus 3 Studied by Immunological and Cytochemical Methods in Electron Microscopy

“…The envelope is not necessary since the virus remains infectious after purification by centrifuging on density gradients which leads to the loss of the envelope [6], The appearance of virus antigens at the cytoplasmic membrane is not sur prising since we have previously shown by the mixed agglutination technique that infected ceils acquired virus-specific surface antigens [5], The use of immunoperoxidase and immunoferritin methods offers the possibility of localizing these antigens at the membrane. Indirect immunological methods, known to be more sensitive than direct methods [20], allowed us to demon strate that the virus-specific acquired antigenicity is strictly localized in the area of the membrane where the virions are budding. Our results were similar, whether peroxidase or ferritin was used as marker.…”

Immuno-Electron Microscope Analysis of the Surface Layers of the Unfertilised Sea Urchin Egg

“…The ferritin-labelled antibody technique of detecting specific antigens is now well proved, having been used for cell surface studies in virus (7)(8)(9)(10), phage (11), bacteria (12), protozoa (13,14), tumour cells (15)(16)(17)(18), and sea urchin gametes (19,20). Intracellular antigens have been studied less, owing to difficulty in making the cell membrane permeable to the large ferritin~,-globulin conjugate without damaging the ultrastructure, but use has been made of complement-plus-antiserum treatment (15,17) or freezing and thawing of the material before applying the labelled antiserum (9,18).…”

Immune Electron Microscopy Using a Two‐layer Method of Ferritin Labelling

“…Although ferritin conjugates still retain their specificity, one of the problems of their use on unfixed cells is that ferritin is seen intracellularly, taken up unspecifically by pinocytosis (Smith & Metzger, 1961). In the sea urchin ferritin is consistently seen in the yolk granules of treated eggs even with ferritin conjugates of normal serum (Baxandall et al, 1962). If, when the indirect method is used, the material is fixed briefly between the application of the unmodified serum and the ferritin anti-y-globulin conjugate the uptake of ferritin by pinocytosis should be prevented (Nachmias & Marshall, 1961) although any unmodified y-globulin which had been pinocytosed in the first treatment (Holter, 1969) will presumably be ferritin labelled.…”

“…Preliminary experiments which have been made on the surface antigens of sea urchin eggs (Baxandall et al, 1962;Perlmann, 1959) demonstrate the much stronger staining produced by the indirect method. The conjugates were made with m-xylylene di-isocyanate as the intermediate coupler, and they were not purified of unconjugated ferritin and y-globulin.…”

“…Generally a ferritin conjugate of the specific antiserum under consideration is applied directly to living or h e d material which is then washed and embedded for electron-microscopical examination. In the present investigation of the surface antigens of sea urchin gametes (Baxandall et al, 1962), however, an alternative method of ferritin labelling has also been employed. This is a two-layer or indirect technique which has been adopted from fluorescence immunology (Coons, 1956).…”