Principles of Fluorescence Spectroscopy

662 FLUORESCENCE SENSING
Figure 19.76. Time-dependent changes in polarization upon mixing
of antibody to cortisol or nonspecific antibody (∆) and fluoresceinlabeled
cortisol and upon addition, at 60 minutes, of 100 ng of unlabeled
cortisol. The antibody is more dilute from top to bottom.
Revised from [292].
the highest due to binding of antibody to Cor-Fl. Free cortisol
from the sample will displace Cor-Fl from the antibody.
The Co4Fl is now free to rotate, and the anisotropy
decreases.
Typical data for a cortisol FPI are shown in Figures
19.76 and 19.77. Cor-Fl was prepared by reaction of cortisol-21-amine
with fluorescein isothiocyanate (FITC). Upon
mixing anti-Cor antibody (Ab) to cortisol with Cor-Fl the
polarization increased, which was presumed due to specific
Figure 19.77. Cortisol fluorescence polarization immunoassay.
Revised from [292].
binding of Ab to Cor-Fl. The specificity of the reaction was
confirmed by adding unlabeled cortisol, which resulted in a
decrease in polarization, and also by the absence of a
change in polarization due to nonspecific antibody (∆).
To perform the cortisol assay one uses a mixture of Ab
and Cor-Fl, to which is added the serum sample. As the concentration
of serum cortisol is increased, the polarization
decreases (Figure 19.77). The polarization values are used
to determine the cortisol concentration. Similar FPIs have
been developed for a wide range of low-molecular-weight
analytes, including antibiotics, 293–294 cocaine metabolites,
295–296 therapeutic drugs, 297–298 the immunosuppressant
cyclosporin, 299–300 and phosphorylated proteins. 301–303
Numerous FPIs are routinely performed on automatic clinical
analyzers. 304
FPIs have advantages and disadvantages. FPIs do not
require multiple antigenic sites, as is needed with heterogeneous
capture immunoassays or RET immunoassays. FPIs
can be performed in a homogeneous format, and may not
require separation steps. However, because FPIs are usually
performed with fluorescein, they are generally limited to
low-molecular-weight analytes. This is because the emission
must be depolarized in the unbound state, which would
not occur for higher-molecular-weight fluorescein-labeled
proteins.
The limitation of FPIs to low-molecular-weight analyte
is illustrated by the FPI for creatine kinase-BB. Creatine
kinase is a dimer, and the subunits can be from muscle (M)
or brain (B). Creatine kinase MB is used as a marker for
cardiac damage, and the presence of CK-BB in the blood
may reflect a number of disease states, including brain trauma.
305–307 Figure 19.78 shows an FPI for CK-BB. 307 In this
particular case the protein was labeled with dansylaziridine
(DANZA), instead of fluorescein. The immunoassay was
also performed with other probes (Table 19.6). One notices
Table 19.6. Fluorescein Polarization Immunoassay
of Creatine Kinase BB a
Polarization
Fluorophore-CK τ (ns) No antibody With antibody
CPM-CKb ~5 0.337 0.342
IAF-CK ~5 0.333 0.339
DNS-CK ~15 0.181 0.224
DANZA-CK
aFrom [307].
~15 0.170 0.242
bCPM, 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin;
IAF, 5'-iodo-acetamidofluorescein; DNS, dansyl chloride; DANZA,
dansylaziridine.