Variations on the staining method in quantitative indirect immunofluorescence assays for Bacillus spores, and the use of fluorescein--protein A.

Quantitative indirect immunofluorescence assays for B. anthracis and B. cereus spores fixed on multispot microscope slides have been performed using a microfluorometer to measure the fluorescence of individual bacteria. A study has been made of variations of the indirect assay sequence, in which the washing operation between application of 1st and 2nd antibody types was omitted. In one modification the addition of the indirect antibody was deferred, and in the second the direct and indirect reagents were added simultaneously to the spores at the start of the incubation period. This simultaneous addition method shows promise for wide application in diagnostic serology. Evidence is presented that fluorescein-protein A (F-PA) can be successfully substituted for fluorescein/sheep anti-rabbit antibody (F-SAR) in the indirect spore assay, and that it is far more active than the F-SAR on a weight basis. About twice as many F-PA molecules as F-SAR molecules are bound to the spore at saturation.