Principles of Fluorescence Spectroscopy

660 FLUORESCENCE SENSING
Figure 19.72. Competitive time-resolved immunoassay for sulfamethazine
(SMZ). Reprinted with permission from [280]. Copyright ©
2004, American Chemical Society.
gated on following decay of the prompt autofluorescence.
Because the lanthanides display millisecond lifetimes, they
continue to emit long after the ns interferences have
decayed. The signal is integrated for a period of time, and
the assay is based on measurement of integrated intensity,
not a decay time. Hence, the phrase "time-resolved" should
not be confused with a lifetime measurement.
Time-resolved immunoassays continue to be developed
for a variety of analytes. 277–281 One example is a competitive
immunoassay for the sulfa antibiotic sulfamethazine
(SHZ) in food. The presence of residual antibiotics
in food is of concern because of the potential health risk to
humans and the development of antibiotic resistance in bacteria.
This immunoassay is based on antibodies against
SHZ. These antibodies are bound to streptavidin-coated
microwell plates (Figure 19.72). These antibodies bind an
analogue of SHZ that contains a covalently bound europium
chelate (SHZ-Eu). This analogue is allowed to bind to
the antibodies. The assay is performed by adding the sample
to the well, allowing time for binding, washing the
plate, then adding the enhanced solution and measuring the
time-delayed emission. The intensity decreases as the SHZ
increases because SHZ displays SHZ-Eu, and the SHZ-Eu
is washed out of the well prior to addition of the enhanced
solution.
19.14.3. Energy-Transfer Immunoassays
Resonance energy transfer provides an obvious approach to
measuring antigen–antibody association, and was suggested
for immunoassays in 1976. 282 Such an assay would typically
be performed in a competitive format, and can be
homogeneous (Figure 19.73). Suppose the analyte is the triazine
herbicide simazine (SZ), which is shown as the open
shape in Figure 19.73. An antibody against SZ is labeled
with the donor Cy5 and bound to the bottom of the well. 283
The acceptor Cy5.5 is bound to BSA, which also contains a
covalently bound analogue of SZ. The assay is then per-
Figure 19.73. Competitive RET immunoassay for the herbicide
simazine. Revised and reprinted with permission from [283].
Copyright © 2001, American Chemical Society.