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Evaluation of Fluorescein Isothiocyanate-labeled Whole Antiserum in the Immunofluorescent Identification of Microorganisms

^a Immunology and Bacteriology Branch, Armed Forces Institute of Pathology, Washington, D.C. 20305

ABSTRACT

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 µg/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 µg of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 µg of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.

¹ Present address: Department of Biology, Wichita State University, Wichita, Kan. 67208.