Albert H. Coons

Improvements in a method for the specific microscopic localization of antigen in tissue cells are described. This method employs antibody labelled with fluorescein isocyanate as a histochemical stain, the specific antigen-antibody precipitate being made visible under the fluorescence microscope. Two isomeric series derived from nitrofluorescein are described.

A method for the specific histochemical demonstration of antibody in cells and parts of cells is described. It consists of carrying out a two stage immunological reaction on frozen sections of tissues: (a) allowing reaction between antibody in the tissue and dilute antigen applied in vitro, and (b) the detection of those areas where this antigen has been specifically absorbed by means of a precipitin reaction carried out with fluorescein-labelled antibody. Examination under the fluorescence microscope reveals the yellow-green fluorescence of fluorescein over those areas where a precipitate has formed. A study of the hyperimmune rabbit on the first few days after the last of a series of intravenous antigen injections reveals that antibody against human gamma-globulin or ovalbumin is present in groups of plasma cells in the red pulp of the spleen, the medullary areas of lymph nodes, the submucosa of the ileum, and the portal connective tissue of the liver. Because of extensive non-specific reactions, the bone marrow could not be examined. Small amounts of antibody were occasionally visible in cells in the lymphoid follicles of the spleen and lymph nodes, so that a minor contribution by lymphocytes to antibody synthesis cannot be excluded.

SummaryA β-anthryl-carbamido derivative of antipneumococcus III rabbit antibody retains the original immunological properties while rendering Type III pneumococci specifically fluorescent in ultraviolet light.It is a pleasure to acknowledge the constant advice and help so generously given by Dr. John F. Enders and Dr. Allan L. Grafflin, and the kind interest shown by Dr. Louis F. Fieser.

SummaryTissue culture preparations infected with agents originally derived from the eruptive lesions of cases of varicella and herpes zoster, as well as control preparations infected with the virus of herpes simplex, were studied by a modification of the fluorescent antibody technic. Employing the infected preparations as antigen, fixation of antibody from human sera derived from cases of varicella, herpes zoster or herpes simplex was detected by the use of a fluorescent antihuman gamma globulin conjugate. Antibody reacting with the varicella and herpes zoster antigens to an almost identical degree appeared during convalescence in serum specimens derived either from cases of varicella or from cases of herpes zoster. Antibody reacting with herpes simplex virus was demonstrated uniformly only in a group of sera derived from cases of recurrent herpes simplex. Immunologic evidence was thus obtained to support the thesis that the etiologic agents of varicella and herpes zoster have been isolated and propagated in vitro.

Abstract The object of this investigation has been to determine the feasibility of using chemically labelled antibodies as reagents for the detection and orientation of antigenic material in mammalian tissue. Such a method requires the retention of specificity by the antibody-molecule during and after the necessary chemical manipulation, a stable chemical linkage between the antibody and its label, and a label that can be detected when present in minute quantities. A further important requirement demands the separation of the labelled-antibody solution from unconjugated tracer-material. In addition a method of this sort would obviously be more useful for many studies if it were possible to determine not only those organs, but those cells which contain the antigen in question. Accordingly we investigated the possibility of employing materials for labelling that could be detected by optical rather than by analytic or radiographic methods.