MODIFICATION OF THE MYCOBACTERIUM-BOVIS EXTRACELLULAR PROTEIN MPB70 WITH FLUORESCEIN FOR RAPID DETECTION OF SPECIFIC SERUM ANTIBODIES BY FLUORESCENCE POLARIZATION

The principle of fluorescence polarization described by Perrin (F. Per rin, J. Phys, Radium 7:390-401, 1926) was applied to the development o f a novel assay that used fluorescein-labeled Mycobacterium bovis secr etory protein MPB70 for rapid detection of anti-MPB70 antibodies in se lected sera from three M. bovis-infected species (elk, Ilama, and biso n). Labeling of purified MPB70 with fluorescein isothiocyanate resulte d in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluor esced strongly with an emission maximum at 518 nm when excited with li ght of a wavelength near 493 nm, and its immunoreactivity with anti-MP B70 monoclonal antibody 4C3/17 was not altered by modification with fl uorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0 .05% lithium dodecyl sulfate, which prevents the occurrence of some no nspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as d etected by enzyme-linked immunosorbent assay (ELISA), reacted with flu orescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninf ected sera (167 to 178 mP), which were close to that obtained in the a bsence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to de tect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology an d its general applicability to the diagnosis of infectious diseases ar e discussed.