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

Abstract

Portions of a whole antiserum to Histoplasmna capsdlaituln were recacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 ,ug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 ,ug 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,ug 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.

Biochemical analysis

“…Fluorometric assays were proposed for 6-mercaptopurine in serum (202), xanthine oxidase (258), and urea in nanoliter samples (527). Fluorescein isocyanate was used to label antiserum for determining and identifying microorganisms (614). A versatile device was proposed for microscopic spectrofluorometry to study excitation, emission, and absorption spectra of nerve cells (621).…”