Measurement of Streptococcal Antigen Synthesis with Fluorescent Antibody. ∗

Abstract

Summary and conclusion

The amount of specific antibody adsorbed by streptococci during the process of de novo somatic antigen synthesis was a function of the amount of antigen on the cell. The fluorescent technic when compared with acid extraction demonstrated that considerably fewer cells were required to observe the progress of synthesis and, in addition, there was obtained a value more quantitative than the estimation of the degree of precipitation in capillary tubes.

Controls with fluorescein-conjugated normal globulin indicated the degree of nonspecific binding of conjugated protein by the streptococcal cells. The basal limit of nonspecific adsorption was considerably higher when fluorescein-labeled whole sera were employed rather than the globulin fractions.

Standard curves obtained with buffered fluorescein solutions were linear within the total range of the instrument. These values were between 0.02 and 2.0 μg of fluorescein per ml. The fluorescent values of Fig. 1 were within a range of 0.04 to 0.25 μg of fluorescein per ml.

An alternative method proved effective. This was the “sandwich” technic of Weller and Coons(7) in which the cells were first incubated with specific antibody and then reacted with fluorescein-conjugated goat anti-rabbit globulin (Microbiological Associates). However, comparatively large amounts of the latter reagent were required to attain a saturation plateau.

It is conceivable that the aforementioned technics with streptococci may be applicable to other systems where quantitative data are desired with limited amounts of particulate antigenic materials.

Summary. A technic is described for fluorometric estimation of the somatic M antigen of group A streptococci. The amount of fluorescein-conjugated antibody adsorbed and eluted from cells was a function of the amount of antigen present during the process of antigenic protein synthesis.