Advanced Techniques in Diagnostic Microbiology

50 Y. F. Wang
by a carbodiimide conjugation method (Staros et al., 1986) such as 1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide EDC coupling method to beads of uniform
size, which are colored with different amounts of red and orange fluorescent dyes
(in a unique ratio) to allow for discrimination based on the relative emission intensities
at the wavelengths of the two fluorescent dyes. Currently, there are 64
different ratios of red and orange fluorescence, which identify 64 distinctly colored
sets of microspheres. Differently colored microsphere sets can be individually
coupled via the surface carboxylate moiety to a specific probe for a unique target.
The flow cytometer analyzes individual microspheres by size and fluorescence,
simultaneously distinguishing three fluorescent colors: green (530 nm), orange
(585 nm), and red (>650 nm). Microsphere size, determined by 90-degree light
scatter, is used to eliminate microsphere aggregates from the analysis. All fluorescent
molecules are labeled with a green-emitting fluorophore. Any green-emitting
fluorochrome can be use as a reporter; however, each fluorochrome has a characteristic
emission spectrum, requiring a unique compensation setting for spillover
into the orange fluorescence channel.
Antigen-conjugated microspheres are added to the well, in the sample, as well
as the fluorescein-conjugate [red-phycoerythrin (R-PE) through biotin and streptavidin]
antispecies detector or secondary antibody (Jones et al., 2002). The red
laser excites specific dyes to identify the analyte [red and orange fluorescent dyes
(detected by FL2/FL3]; the green laser excites a different dye to quantify the result
(a green fluorescent reporter dye FL1) (Vignali, 2000; Mandy, 2001). The
fluorescence emission of each bead of the specific antigen was determined with a
fluorescence-activated cell scanner (FACScan, Becton-Dickinson, San José, CA,
USA), a benchtop flow cytometer (multiparameter flow cytometer that is based on
a single 488-nm excitation laser), with FlowMetrix hardware for data acquisition
and analysis (Luminex Corp., Austin, TX, USA). The software allows rapid classification
of microsphere sets on the basis of the simultaneous gating on orange
and red fluorescence.
The Luminex instrument is a dual-laser flow analyzer. The first laser excites
the fluorochrome mixture intrinsic to the microspheres, enabling the bead identity
to be determined as the beads pass single file through the laser path in the flow
cell. The second laser excites the extrinsic fluorochrome (R-PE) that is covalently
attached to the secondary antibodies. The dual lasers allow the operator to mix
beads with different antigens together in a well of a filter plate, thus enabling
multiplex analysis of different antibody specificities at one time. Orange and red
fluorescence are used for microsphere classification, and green fluorescence is
used for analyte measurement (Fulton et al., 1997).
Contrast of These Techniques
The contrast with immunoassay techniques is shown in Table 4.1.
ELISA assays are relatively inexpensive, can be adapted for high-throughput use,
and thus are commonly used in research and clinical laboratories. The enzymes