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Complexes of Haemophilus influenzae cells with and without intact capsular material were prepared with type specific antisera. Thin sections of positively stained specimens were prepared and examined in the electron microscope. In the absence of capsular material the antibodies were observed bound close to one another in the immediate area of the bacterial cell wall. When capsular antigen was present the antibodies were farther separated from one another in the complex and appeared to be attached at random sites along the capsular polymers with occasional individual particles being observed. This positive staining approach appears to be a useful method to observe the molecular structure of precipitated immune complexes. [ABSTRACT FROM AUTHOR]

A simple test system is described, for the demonstration of antigen-antibody reactions capable of causing eosinophil granule lysis in vitro. The antigen preparations used were extracts of the nematode Amplicaecum robertsi and body fluid of Ascaris suum. Antisera were obtained from rats infested with Amplicaecum. Eosinophils were obtained from the peritoneal cavity of normal rats. Centrifugation of the cells to form a cell button was an essential step in the procedure. Lysis of eosinophils occurred with antiserum obtained from the animals between the 12th and 32nd days of infestation with Amplicaecum, and was accompanied by vacuole formation in macrophages and mast cell disruption. The reaction was most pronounced during the 3rd week. Serum from adrenalectomized infested animals caused the most marked changes in eosinophils. Serum from cortisone- treated infested animals failed to cause eosinophil changes. Attempts at purification of the antigen in Ascaris body fluid resulted in two fractions with marked activity in the test system. The same two fractions were found to form precipitin lines on agarose gel diffusion against rat antiserum. It is postulated that antigen-antibody complexes soluble in low concentration were responsible for the changes observed in the eosinophils, macrophages and mast cells. One or more labile factors in the serum were found to be necessary for eosinophil granule lysis. The evidence, though incomplete, would favour the suggestion that both labile antibody and complement were necessary. [ABSTRACT FROM AUTHOR]

Antigen-antibody precipitates, prepared either in solution in antibody excess or by cutting precipitin lines from immunoelectrophoretic plates have been shown to bind specifically to the homologous antigen in tissue sections. This finding has been used as the basis of an immunofluorescent technique in which antigen-antibody precipitates are applied to a section and then stained with a fluorescein-conjugated antiglobulin reagent. The technique allows the localization of antigens in tissue even where they are not characterized or purified and where monospecific antisera are not available. [ABSTRACT FROM AUTHOR]

Immune complexes composed of two insoluble and two soluble antigens and their corresponding antibodies of rabbit origin were separated by electrophoresis in agar gel performed at 56°. Antibodies could then be demonstrated in the cathodal part of the electrophoretic field; they formed precipitation lines with goat antiserum to rabbit serum. The separation of soluble antigens could be demonstrated by the precipitation reaction with their corresponding antisera. [ABSTRACT FROM AUTHOR]

Using the criteria of generalized anaphylaxis, the Schuhz­Dale test, and mast cell degranulation occurring on contact with antigen in vitro, a study has been made of the ability of antisera, prepared in various species, to sensitize guinea pig tissues passively. Whereas small amounts of guinea pig and rabbit antibodies and rather larger amounts of monkey, dog and human antibodies were effective, antibodies from goat, horse, rat, fowl and a human auto-immune thyroid antiserum were ineffective. These results are in general agreement with those in the literature. Using antibodies labelled with 131I, no gross differences were found between those which sensitized and those which did not, either in respect of uptake on guinea pig mesentery in vitro, or retention in the tissue after uptake in vivo. By means of antigen labelled with 131I it was also shown that the antibody adsorbed on guinea pig mesentery could combine equally well with antigen, whether the antibody came from rabbit (sensitizing) or rat (non-sensitizing). These experiments also indicated that the amount of adsorbed antibody necessary for sensitization is very small. Reversed passive anaphylaxis was studied with a variety of different antigen-antibody systems, using the criteria listed above. It occurred when, but only when, the antigen was a γ-globulin, and when both antigen and antibody globulins came from species whose antibodies were able to sensitize guinea pigs for direct passive anaphylaxis. It is concluded that the explanation of the differences between antibodies or antigens which sensitize guinea pigs passively, and those which do not, is to be found in the nature of the antigen-antibody combination as well as in the manner of ‘fixation’ on the tissues. The possible importance of ability to activate guinea pig complement is discussed. [ABSTRACT FROM AUTHOR]