Chauncey W. Smith

Superiority of fluorescein isothiocyanate (Riggs) for fluorescent-antibody technic with a modification of its application.

Summary 1) The globulin fractions of antisera representing bacterial, viral, mycotic agents and antiglobulin fractions were labeled with 2 derivatives of fluorescein amine by 3 methods. 2) Fluorescein isothiocyanate was shown to be superior in stability, ease of conjugation, and degree of fluorescence. This direct method of adding the dye to a dilute buffered antiserum eliminates the need for organic reagents which may denature the protein.

Use of Contrasting Fluorescent Dye as Counterstain in Fixed Tissue Preparations

Summary A counterstaining method is described that gives a contrasting reddish-orange background when used with fluorescein-labeled antibody systems. It curtails non-specific fluorescence in tissues and tissue cultures. The possibility of a nonspecific protein-protein reaction is discussed. This reaction apparently plays no part in the serological system to which it has been added.

Immune Electron Microscopy

Summary A method is described by which immune systems can be studied at subcellular level with the electron microscope, using a ferritin-antibody conjugate. A conjugated immune serum to Staphylococcus aureus was applied to homologous organisms, embedded in methacrylate with uranium and thin sections cut. An antigen-antibody reaction was detected by attachment of the ferritin molecule in the capsule-like material. Use of formalin in primary fixation for electron microscopy as well as nonspecific immunofluorescence is discussed.

Identification of Pathogenic Fungi in Surgical and Autopsy Specimens by Immunofluorescence.

A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.

Identification of Listeria monocytogenes by the Fluorescent Antibody Technic

Summary Preparation is described of a polyvalent somatic fluorescein labeled antiserum which was specific for 30 strains of Listeria tested but did not react with 180 heterologous strains. Preparation and specificity of flagellar and whole cell antigens are discussed as well as some technical implications of fluorescent antibody technic.

Demonstration of Venezuelan equine encephalomyelitis in tissue culture by immunofluorescence.

Summary In a guinea pig heart cell line, infected with an egg-adapted strain of Trinidad VEE, the virus could be demonstrated by immunofluorescence prior to development of CPE. Formalin fixation preserved the antigenicity, and the cover slips could be stored in this fixative at 4°C for periods up to 2 weeks without diminution of specific fluorescence. A whole burro serum conjugated with fluorescein isothiocyanate with a counterstain added proved a more reliable reagent than fractionated or absorbed sera. The model as described has the possibility of obtaining a specific viral diagnosis at the clinical level in the period normally required for initial isolation by conventional cell culture technics.

Serologic typing of Listeria monocytogenes by gel diffusion using thermostable antigens.

Summary A procedure is described by which acid extracts of capsularenhanced L. monocytogenes are serotyped by gel diffusion. Sixty strains were placed in 5 serotypes that coincide with the serotypes obtained by conventional methods using somatic “O” and flagellar “H” factor sera. The procedure has been simplified so that it can be efficiently utilized by facilities other than specialized typing centers. We are indebted to Dr. Milton M. Gray for confirming serotypes of cultures and advice during this investigation.