Advanced Techniques in Diagnostic Microbiology

4. Advanced Antibody Detection 49
UV light is used much less in antibody detection than in antigen detection and will
not be covered as well. However, IFA techniques such as those used in TRF, FC,
and xMAP (Table 4.1) are discussed below.
TRF assays use a lanthanide chelate such as europium (Eu 3+ ) or samarium
labels. These labels have unique properties such as a long fluorescence decay
time so to lower background interference. TRF is similar to ELISA, except that
the capture antigen affixed to the solid phase is mixed with the test sample, and
the complex if any is mixed with diluted detector antibody that is labeled with
lanthanide chelate. A low-pH enhancement solution added can cause lanthanide to
dissociate from the labeled compound and is highy fluorescent (Aggerbeck et al.,
1996; Peruski et al., 2002).
TRF exploits the differential fluorescence life span of lanthanide chelate labels
compared with background fluorescence. The labels have an intense long-lived
fluorescence signal and a large Stokes shift, resulting in assays with a very high
signal-to-noise ratio and excellent sensitivity (Hemmila et al., 1984). TRF produces
its signal through the excitation of the lanthanide chelate by a specific wavelength of
light. Fluorescence is initiated in TRF with a pulse of excitation energy, repeatedly
and reproducibly.
FC is a commonly used IFA. The first use of flow cytometry for analysis of
microsphere-based immunoassays was published in 1977 (Horan and Wheeless,
1977; McHugh, 1994). Initially, different-sized microspheres were used for simultaneous
analysis of different analytes (Horan and Wheeless, 1977). A fluorescent
probe is added to a liquid suspension with sample, which is then streamed past a
laser beam where the probe is excited. A detector analyzes the fluorescent properties
of the sample as it passes through the laser beam. Using the same laser
excitation source, the fluorescence may be split into different color components so
that several different fluorophores can be measured simultaneously and analyzed
by specialized software. A flow cytometer has the ability to discriminate different
particles on the basis of size or color, thus making the multiplexed analysis possible
with different microsphere populations in a single tube and in the same sample at
the same time.
xMAP is definitely an emerging antibody detection method and has been referred
to as, multiplexed particle-based flow cytometric assays technology, fluorescent
microsphere immunoassay (MIA), fluorescence covalent microbead immunosorbent
assay (FCMIA), multiplexed indirect immunofluorescence assay, or multiplex
flow cytometry. This two-step suspension method is based on fluorescent detection
using the FlowMetrix analysis system (Fulton et al., 1997). Systems using xMAP
technology perform assays on the surface of color-coded beads (microspheres)
that are covered with capture antigens that react with the target antibodies. The
microbeads have surface-binding characteristics and a dyeing process to create up
to 100 unique dye ratios, which are used to identify an individual microsphere in
a single well.
Specific dyes permeate the polystyrene microspheres that are 5.5 μm indiameter
and are composed of polystyrene and methacrylate to provide surface
carboxylate functional groups on the surface. Each antigen is covalently linked