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Oak Ridge Conference |
1
University of Texas Southwestern Medical Center, 5325 Harry Hines Blvd., Dallas, TX 75235.
a Address correspondence to this author at: Luminex Corporation, 1638 Osprey Dr., DeSoto, TX 75115. Fax 972-224-9689;
Abstract
The FlowMetrixTM System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95®-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.
The first use of flow cytometry for analysis of microsphere-based immunoassays was published in 1977 (1), and recently reviewed by McHugh (2). Because a flow cytometer has the ability to discriminate different particles on the basis of size or color, the possibility of multiplexed analysis with different microsphere populations is suggested. Multiplexed analysis is the ability to perform multiple discrete assays in a single tube with the same sample at the same time. The use of different-sized microspheres for simultaneous analysis of different analytes was originally proposed by Horan et al. (3). Two distinct sizes of microspheres were used for simultaneous detection of two different antibodies by flow cytometry, and subsequently expanded to the use of four different sizes of microspheres to detect four different specificities of anti-HIV antibodies (4)(5). Size discrimination of microspheres allows simultaneous detection of small numbers of analytes, but the inability to distinguish aggregates of smaller microspheres from larger microspheres severely limits the extent of multiplexing that can be achieved. In contrast, differential dyeing of identically sized microspheres with two different dyes, emitting in two different wavelengths, allows aggregates to be distinguished and permits discrimination of at least 64 different sets of microspheres.
The FlowMetrixTM system (Luminex Corp.) performs multiplexed analysis of up to 64 different reactions simultaneously by using a flow cytometer and digital signal processor to perform real-time analysis of multiple microsphere-based assays. The three major components of the system are a benchtop flow cytometer, microspheres, and computer hardware and software. The flow cytometer analyzes individual microspheres by size and fluorescence, distinguishing three fluorescent colors—green (530 nm), orange (585 nm), and red (>650 nm)—simultaneously. Microsphere size, determined by 90-degree light scatter, is used to eliminate microsphere aggregates from the analysis. Orange and red fluorescence are used for microsphere classification, and green fluorescence is used for analyte measurement. The FlowMetrix system is currently configured for the Becton Dickinson FACScan®, a multiparameter flow cytometer that is based on a single 488-nm excitation laser (Becton-Dickinson Immunocytometry Systems).
Microspheres serve as the vehicles for molecular reactions. The
microspheres are 5.5-µm polystyrene microspheres that bear
carboxylate functional groups on the surface. The microspheres are
available in 64 distinct sets that are classified by the flow cytometer
by virtue of the unique orange/red emission profile of each set
as shown in Fig. 1
. The microspheres can be covalently coupled to virtually any
amine-containing "target" molecule through surface carboxylate
groups; alternatively, avidin-coupled microspheres can be used for
binding biotinylated molecules. Microspheres of this size provide
sufficient surface area for covalent coupling of 1–2 x
106 target molecules per microsphere. In addition, the
small size allows the microspheres to remain in suspension for several
hours, which is more than sufficient for assay setup and analysis, and
also provides near-fluid-phase reaction kinetics.
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FlowMetrix hardware and software provide complete control of the flow cytometer and perform real-time classification of the microspheres and analysis of the microsphere-based reactions simultaneously. The hardware is a personal computer interface card that provides communication between the computer and the FACScan. The interface card has an on-board, high-speed digital signal processor that is capable of performing >30 million mathematical functions per second. The interface card connects to the computer interface of the FACScan through a switch, allowing alternate use of the FlowMetrix system or the standard computer system. The software is a Windows95®-based 32-bit application that provides a "multiplexed mode" for automated multiplexed analysis, as well as a "data acquisition mode" for nonautomated gating and data acquisition. Both modes automatically record captured data to flow cytometry standard, FCS 2.0, data files for use with third-party data analysis tools. In addition, statistical analysis generated by the software is recorded to comma-separated-value files that can be read by third-party spreadsheet programs.
Fluorescent antibodies, antigens, or nucleic acid probes provide specific signals for each reaction in a multiplexed assay. Because each fluorescent reactant binds specifically to a target that is present on only one bead set in a multiplexed assay, the soluble reactants do not need to be differentially labeled. All fluorescent molecules are labeled with a green-emitting fluorophore such as Bodipy® (Molecular Probes) or fluorescein isothiocyanate. 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.
To prepare a multiplexed assay, individual sets of microspheres are conjugated with the target molecules required for each reaction. Target molecules may be antigens, antibodies, oligonucleotides, receptors, peptides, enzyme substrates, etc. A fluorescent reactant is prepared for each target molecule. Fluorescent reactants may be oligonucleotides, antigens, antibodies, receptors, etc., i.e., any molecule that will bind to the target molecule. After optimizing the parameters of each assay separately in a nonmultiplexed format, the assays can be multiplexed by simply mixing the different sets of microspheres. The fluorescent reactants also are mixed to form a cocktail for the multiplexed reactions. The microspheres are then reacted with a mixture of analytes, such as a serum sample, followed by the cocktail of fluorescent reactants. After a short incubation period, the mixture of microspheres, now containing various amounts of green fluorescence on their surfaces, are analyzed with the flow cytometer. Data acquisition, analysis, and reporting are performed in real time on all microsphere sets included in the multiplex. As each microsphere is analyzed by the flow cytometer, the microsphere is classified into its distinct set on the basis of orange and red fluorescence, and the green fluorescence value is recorded. One hundred individual microspheres of each set are analyzed and the mean value of the green fluorescence is reported. In the present report, an immunoassay for allergen-specific IgE and IgG testing, and DNA hybridization with sequence-specific oligonucleotides for genetic testing are presented. Both assay systems take advantage of the multiplexing capabilities of the system by performing 16 simultaneous analyses in the same reaction.
Materials and Methods
Microspheres.
Carboxylate-modified polystyrene
microspheres (5.5 µm diameter) were dyed with various amounts of
orange-emitting and red-emitting fluorochromes (Luminex Corp.). Eight
different concentrations of each of the two fluorochromes were mixed in
all 64 possible combinations and the mixtures were used to prepare 64
microsphere sets with unique orange-red emission profiles (Fig. 1
).
Thus, the flow cytometer measures both the color, or emission
wavelength, and the intensity of each dye. The first 16 microsphere
sets produced were used in this study.
Reagents.
Grass allergen extracts, canine sera, and
affinity-purified goat anti-canine IgE were kindly provided by Bill
Mandy, BioMedical Services. Rabbit anti-goat IgG–fluorescein
isothiocyanate (FITC), goat anti-canine IgG-FITC,
2-(N-morpholino)ethanesulfonic acid (MES), and bovine serum
albumin (BSA) were purchased from Sigma Chemical
Co.1
1-Ethyl-3-3(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC) and N-hydroxysulfosuccinimide
(sulfo-NHS) were purchased from Pierce Chemicals.
Synthetic oligonucleotides.
Sixteen oligonucleotides
corresponding to allelic sequences within the second exon of the
HLA-DQA1 gene and their corresponding complementary sequences were
synthesized with standard automated synthesis techniques (Oligos,
Etc.). The oligonucleotides were modified at the 5' end during
synthesis with an amino-linker. The amino-linker consists of an amino
group attached to a chain of six carbon–carbon linkages. Before the
addition of the amino-linker, a chain comprising nine carbon–carbon
and aliphatic ether linkages was introduced as a spacer.
Double-stranded (ds) oligonucleotides used as competitors in
hybridization experiments were prepared by annealing equal amounts of
unlabeled complementary oligonucleotides.
Allergen-coated microsphere preparation.
Sixteen
different grass allergens were conjugated to 16 different microsphere
sets through their surface carboxylate groups with a carbodiimide
coupling method. Twenty microliters of 4 g/L microspheres (~8 x
106 microspheres) were activated in 100 µL of 50 mmol/L
sodium phosphate buffer, pH 7.0, containing 500 µg of EDC and 500
µg of sulfo-NHS. Microspheres were washed twice with 100 µL of PBS,
pH 7.4, with centrifugation at 13 400g for 30 s to
harvest the microspheres. Activated, washed microspheres were suspended
in 50 µL of diluted allergen extract (1:100 in PBS, pH 7.4). After
2 h, allergen-coated microspheres were washed twice with 100 µL
of 0.2 mL/L Tween 20, 1 g/L BSA in PBS pH 7.4 (PBSTB), suspended in 1
mL of PBSTB, and counted on a hemocytometer. The concentration of
microspheres was adjusted with PBSTB to 3 x 109/L and
the suspensions stored at 2–8 °C.
Multiplexed assay for grass allergen-specific IgE.
Equivalent amounts of each of the 16 grass allergen-loaded microspheres
were mixed. Twenty microliters of the mixture (~60 000 microspheres
total) were mixed with 60 µL of a 1:3 dilution of canine serum in
PBSTB, and the mixture was incubated for 30 min. Microspheres were
washed in 200 µL of PBSTB and suspended in 40 µL of a 50 mg/L
solution of goat anti-canine IgE. After incubation for 30 min, beads
were washed in 200 µL of PBSTB and treated with 40 µL of rabbit
anti-goat IgG–FITC at 20 mg/L in PBSTB. After 30 min, the assay was
diluted to 300 µL with PBSTB and assayed with the FlowMetrix system.
Negative controls included the microspheres with canine serum, without
the goat anti-canine IgE and with the rabbit anti-goat IgG–FITC.
Allergen-specific canine IgE was determined by subtracting the mean
fluorescence intensity (MFI) of the green channel for the negative
controls for each grass allergen from the MFI for the tubes including
the goat anti-canine IgE.
Multiplexed assay for grass allergen-specific IgG.
Twenty microliters (60 000 microspheres) of the microsphere mixture
were reacted with 20 µL of a 1:10 dilution of canine serum in PBSTB,
and the mixture was incubated for 30 min. Microspheres were washed in
200 µL of PBSTB and suspended in 25 µL of a 50 mg/L solution of
goat anti-canine IgG–FITC. After 30 min, the mixture was diluted to
300 µL in PBSTB and assayed with the FlowMetrix system. Negative
controls included the microspheres with no canine serum and with the
goat anti-canine IgG–FITC. Allergen-specific canine IgG was determined
by subtracting the MFI for the negative control for each grass allergen
from the MFI for the tubes including canine serum.
Coupling of oligonucleotides to microspheres.
HLA-DQA1
allele-specific oligonucleotides corresponding to the noncoding strand
[denoted by (-)] were covalently coupled to unique sets of
carboxylate-modified polystyrene microspheres by using water-soluble
carbodiimide (EDC). Briefly, 100 µL of a 0.001 mol/L solution of
oligonucleotide in 0.1 mol/L MES buffer (pH 4.5) was added to 1.0 mL of
microspheres (1% solids) in 0.1 mol/L MES buffer (pH 4.5). Fifty
microliters of a 10 g/L solution of EDC were added to this mixture, and
the solution was mixed vigorously. After incubation at room temperature
for 30 min, the same amount of EDC was added again and the solution was
mixed. Incubation at room temperature was continued for another 30 min
and then the microspheres were pelleted by centrifugation at
11 750g for 4 min. Microspheres were washed one time in PBS
containing 0.2 mL/L Tween 20, and two times in PBS containing 1 g/L
sodium dodecyl sulfate (SDS) by resuspending the microsphere pellet in
the solution and then pelleting the microspheres by centrifugation as
described. The final microsphere pellet was resuspended in 400 µL of
0.1 mol/L MES buffer (pH 4.5) and stored at 4°C.
Fluorescent labeling of oligonucleotides.
HLA-DQA1
allele-specific oligonucleotides corresponding to the coding strand
[denoted by (+)] were fluorescently labeled with Bodipy FL-X
{6-[(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino]hexanoic
acid, succinimidyl ester} (Molecular Probes). Briefly, a 400-µL
solution containing 0.000002 mol/L oligonucleotide in 0.1 mol/L sodium
bicarbonate and 50 g/L dimethyl sulfoxide (DMSO) (pH 8.2) was mixed
with 30 µL of Bodipy FL-X (10 g/L in DMSO) at room temperature for
16–18 h. The mixture was desalted on a PD-10 column (Pharmacia
Biotech) equilibrated in TE (0.010 mol/L Tris-HCl + 0.001 mol/L EDTA,
pH 8.0) to remove unreacted dye, and stored at 4 °C.
Competitive hybridization assay.
Forty femtomoles of
each fluorescent oligonucleotide and 24 µg of fragmented salmon sperm
carrier DNA were mixed with 500 fmol of ds oligonucleotide in a total
volume of 26.7 µL of reaction buffer (0.050 mol/L KCl, 0.020 mol/L
Tris-HCl pH 8.4, and 0.003 mol/L MgCl2), and the mixture
was heated to 100 °C for 10 min. Twenty-five microliters of
55 °C-equilibrated 2x hybridization buffer (1x hybridization
buffer consists of 2.25 mol/L tetramethylammonium chloride, 1.13 g/L
SDS, 0.00225 mol/L EDTA, and 0.056 mol/L Tris-HCl pH 8.0) were then
mixed with the solution, and the mixture was incubated at 55 °C for
15–30 min. After this incubation, 2.5 µL of the microsphere mixture,
containing 8000 microspheres of each of the 16 sets (128 000
microspheres total) in 2x hybridization buffer, were added to the
mixture and the solution was vortex-mixed. The hybridization mixtures
were incubated for an additional 15 min at 55 °C. Without washing,
samples were diluted to 250 µL with 55 °C-equilibrated
hybridization buffer. Dilutions were performed immediately before
analysis. Simultaneously, competitive hybridizations in the absence of
competitor were performed to determine maximal hybridized
oligonucleotide values.
Flow cytometric analysis.
Assays were performed in a
volume of 50 to 100 µL; however, before analysis each reaction was
diluted to ~300 µL to provide sufficient sample volume for the flow
cytometer. Immediately after dilution, samples were analyzed on a
FACScan benchtop flow cytometer (Becton-Dickinson) with FlowMetrix
hardware and software for operational control of the flow cytometer and
for data acquisition and analysis (Luminex Corp.). At least 100
microspheres were analyzed for each microsphere set. Analysis times
averaged ~20 s. The software allows rapid classification of
microsphere sets on the basis of the simultaneous gating on orange and
red fluorescence. Separate histograms of logarithmic green fluorescence
intensity representing each microsphere set were acquired. The MFI of a
logarithmic green fluorescence histogram was taken as a measure of the
fluorescence associated with the corresponding microsphere set and
defined as the value of hybridized oligonucleotide. Percent inhibition
of hybridization on a microsphere set was determined with the
formula:
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Results and Discussion
To establish the utility of the FlowMetrix system for multiplexed analysis, assays have been developed representing both immunoassays for analysis of serum proteins and DNA hybridization-based assays for genetic analysis.
Immunoassays.
An area of clinical immunology that is
suited particularly well to panel-based analysis is determination of
the serum concentrations of allergen-specific IgE antibodies. In many
instances, allergic individuals are tested for serum IgE and IgG
concentrations specific for a related series of allergens, such as
grasses, trees, or bee venoms; in other cases, panels of geographically
related allergens are examined. Fig. 2
depicts the results of a multiplexed assay for canine IgE and
IgG antibodies specific for 16 different grass allergens. The entire
analysis of each serum sample was performed in two tubes, one for IgE
and one for IgG. Each reaction contained 16 different microsphere sets,
each set coated with a different allergen. After incubation with serum
samples and fluorescent antibodies, the reactions were analyzed with
the FlowMetrix system. Removal of excess fluorescent antibodies is not
required because of the extremely small sensing volume of the flow
cytometer. The analysis time on the flow cytometer is ~15 s for each
sample, and the results are reported, instantaneously, in a spreadsheet
format. The green fluorescent signal for each microsphere set, reported
as MFI, is proportional to the amount of bound antibodies; thus,
appropriate calibrators can be used to perform quantitative assays. The
data shown in Fig. 2
represent the measurement of allergen-specific
antibodies in canine sera; similar principles would apply to
measurement of human antibodies. Serum A96326 demonstrated low IgE
reactivity to most of the grass allergens as compared with serum
A96323. In contrast, serum A96326 contained high concentrations of
allergen-specific IgG, compared with allergen-specific IgG in serum
A96323 for several of the grass allergens. Perhaps the most important
point to be gleaned from these data is that 64 individual measurements
were performed, analyzed, and reported from only four reaction tubes.
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Genetic analysis.
A widely used method for ascertaining
the presence of particular sequences in target nucleic acids is
hybridization with short, synthetic oligonucleotide probes of known
sequence (6). Short oligonucleotides of eight to 20 bp are
used because they display a high degree of hybridization specificity
(7)(8)(9). Molecular hybridization with short
oligonucleotides has been applied to numerous problems in research and
medicine that require DNA sequence analysis. These include detection of
mutations for diagnosis of genetic and malignant diseases
(6)(10)(11) and for classification
of HLA alleles for tissue typing (12). The experiments
described here involved a series of 16 oligonucleotide probes
representing allelic sequences of the human HLA-DQA1 gene (Fig. 3
).
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Hybridization properties of microsphere-attached
oligonucleotides.
Parallel hybridization assays necessitate that a
single reaction condition is used for hybridization of the
oligonucleotide probes to the target nucleic acid. Hybridization
solutions containing tetramethylammonium chloride are useful for this
purpose since tetramethylammonium chloride minimizes the effects of
base composition on duplex association and disassociation rates
(13)(14). The stringency of hybridization is
then controlled strictly as a function of the length of contiguous
complementarity. All hybridization experiments reported here were
performed in the presence of 2.25 mol/L tetramethylammonium chloride.
Before attempting to multiplex the 16 oligonucleotide-substituted
microspheres, the basic parameters of microsphere-attached
oligonucleotide hybridization were examined with a single
microsphere–oligonucleotide. A constant number (8000) of one
oligonucleotide-coated microsphere set, 3403-, was hybridized to
increasing quantities of complementary (3403+) or unrelated (5501+)
fluorescent oligonucleotide at 55 °C for 15 min, and the green
fluorescence associated with the microspheres was measured with the
FlowMetrix system (Fig. 4
A). Microsphere-bound fluorescence increased in a linear manner
up to 40 fmol of input complementary fluorescent probe. Identical
quantities of input noncomplementary fluorescent oligonucleotide showed
little or no hybridization. Each of the 16 DQA1 oligonucleotide pairs
showed similar hybridization properties (data not shown), and all
future experiments were performed with 8000 of each microsphere and 40
fmol of each fluorescent oligonucleotide.
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The kinetics of hybridization of microsphere-bound DQA3403-
oligonucleotide to complementary (DQA3403+) and unrelated (DQA5501+)
fluorescent oligonucleotides is shown in Fig. 4B
. Hybridization of the
microsphere-bound DQA3403- oligonucleotide with its complementary
probe is rapid, requiring no more than 15 min to reach completion.
During this time period, there is little or no hybridization of the
unrelated fluorescent probe. Therefore, a period of 15 min was used
routinely for hybridization of the microsphere-attached
oligonucleotides to the fluorescent oligonucleotide probes
To confirm that hybridization at 55 °C in 2.25 mol/L
tetramethylammonium chloride allowed discrimination between
complementary and closely related 18-bp sequences, hybridization
reactions were performed at various temperatures with fluorescent
probes representing complementary, single, and double mutants, and
unrelated sequences. The DQA3403- microsphere set was selected for
analysis because the DQA3403 sequence differs from DQA3402 by only a
single base and this mismatch is positioned such that the
oligonucleotides have 13 contiguous identical bp, the longest stretch
of identity between any of the 16 oligonucleotides in the HLA-DQA1
panel. As shown in Fig. 4C
, hybridization temperatures <55 °C did
not allow discrimination of the point mutant DQA3402 oligonucleotide
from the complementary sequence. At 55 °C, the point mutant
hybridized with ~50% lower efficiency than the complementary
sequence, and the double mutant and unrelated oligonucleotides did not
hybridize. No hybridization of any oligonucleotides occurred at
65 °C. Thus, hybridization at 55 °C in 2.25 mol/L
tetramethylammonium chloride should allow detection and discrimination
of perfectly complementary oligonucleotide duplexes from mismatched
ones.
DNA sequence analysis by competitive hybridization.
The
purpose of these experiments was to establish a general system for
multiplexed hybridization analysis of DNA sequences that uses
unlabeled, ds target DNA, such as a standard PCR product, as the test
material. The assay system contains 16 different microspheres coupled
to the 16 different DQA1 oligonucleotide target probes, and the 16
complementary fluorescent DQA1 probes (Fig. 3
). In the presence of a
complementary competitor sequence, hybridization between the
complementary fluorescent probe and its complementary microsphere
target will be reduced.
To test the specificity of this system for DNA sequence analysis by
competitive hybridization with unlabeled DNA targets, unlabeled
oligonucleotides were used as model competitors. Preliminary
experiments demonstrated that 500 fmol of ds oligonucleotide resulted
in maximal inhibition of fluorescent probe hybridization and that the
hybridization was complete within 15 min (data not shown). Each
unlabeled ds HLA-DQA1 oligonucleotide was denatured and hybridized to
the mixture of the 16 HLA-DQA1 fluorescent oligonucleotides. Then, the
mixture was hybridized to the mixture of 16 HLA-DQA1 microspheres.
Competition of oligonucleotide duplex formation on microsphere sets
with the HLA-DQA1 oligonucleotide competitors was very effective and
specific (Table 1
). Denatured ds HLA-DQA1 oligonucleotides inhibited
hybridization on complementary microsphere sets 62–93%, and inhibited
highly homologous or unrelated microsphere sets <21%. Thus, the
HLA-DQA1 competitive hybridization test performed accurately with
denatured ds oligonucleotide competitors. This multiplexed
hybridization assay has been used to perform HLA-DQA1 tissue typing of
PCR-amplified human genomic DNA (P. Smith et al., submitted for
publication). The assay types DQA1 alleles in both homozygous and
heterozygous samples, and the entire analysis is accomplished in 30 min
after PCR amplification.
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These studies have demonstrated the ability of the FlowMetrix system to perform highly multiplexed assays for analysis of specific protein–protein interactions, such as immunoassays, and for analysis of specific DNA sequences. The system provides several advantages for analysis of biologically and medically relevant molecules, including speed, economy, and advanced analytical capabilities. The system reduces assay time by performing multiple analyses simultaneously rather than sequentially. The no-wash format of many microsphere-based assays, particularly in the final detection step, is considerably faster than microtiter-based assays that require multiple washing steps to remove excess reagents. In addition, the rapid kinetics of microsphere-based assays allow shorter incubation times than conventional solid-phase assays. The reduced assay time also reduces labor costs for performing multiple analyses. Reagent usage for microsphere-based assays is 10- to 1000-fold less than microtiter-based assays. Multiplexing allows unique analysis of molecular interactions that can only be performed in a multiplexed format. For example, direct comparison of receptor binding avidity for related ligands could be performed under truly competitive conditions where the receptor and all ligand variants are present in the same reaction at the same time.
Most, if not all, assays for biomolecules can be adapted to the FlowMetrix system. Immunoassays have been developed with all assay formats available with microtiter-based technology, including direct binding assays, immunometric or capture/sandwich assays, and competitive inhibition assays. In addition, receptor/ligand analysis and epitope mapping are well suited for the multiplexed format. Multiplexed hybridization of nucleic acids can be used for tissue typing, diagnosis of genetic disease, paternity/forensic testing, tests for oncogenes, tests for foreign pathogens, and tests for mRNA/cDNA expression. Because of the real-time analysis, no-wash format, and solution-phase kinetics of microsphere-based assays, multiplexed enzyme assays also can be performed to analyze both substrate specificity and kinetic data for multiple enzymes or multiple substrates simultaneously. For example, multiple microsphere sets could be prepared, each bearing a different fluorescent peptide sequence. These microsphere sets could then be mixed and used to examine the specificity or relative activity of an endopeptidase for the different substrate sequences. In this case, enzymatic activity would be measured as the loss of fluorescence from the microspheres.
The FlowMetrix system represents a revolutionary new technology that can be applied to virtually any application that requires analysis of molecular interactions, including basic research, clinical diagnostic testing, high-throughput drug screening, environmental testing, and agricultural testing. This system is unique in its ability to provide multiplexed, high-throughput analysis coupled with real-time data analysis. The system offers excellent sensitivity, precision, speed, and economy.
Acknowledgments
This work was supported by Luminex Corporation, Austin, TX. We thank Kerry Oliver for reading and editing the manuscript, and Shawna Kennedy, Christy O'Brien, and Van Fitzgerald for expert technical assistance.
Footnotes
Luminex Corporation, Austin, TX 78727.
1 Nonstandard abbreviations: FITC, fluorescein isothiocyanate; MES, 2-(N-morpholino)ethanesulfonic acid; BSA, bovine serum albumin; EDC, 1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide hydrochloride; ds, double-stranded; NHS, N-hydroxysulfosuccinimide; PBSTB, PBS-Tween-BSA buffer; MFI, mean fluorescence intensity; SDS, sodium dodecyl sulfate; and DMSO, dimethyl sulfoxide. 
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T. B. Martins, R. Burlingame, C. A. von Muhlen, T. D. Jaskowski, C. M. Litwin, and H. R. Hill Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens Clin. Vaccine Immunol., November 1, 2004; 11(6): 1054 - 1059. [Abstract] [Full Text] [PDF]
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J. S. Klutts, R. S. Liao, W. M. Dunne Jr., and A. M. Gronowski Evaluation of a Multiplexed Bead Assay for Assessment of Epstein-Barr Virus Immunologic Status J. Clin. Microbiol., November 1, 2004; 42(11): 4996 - 5000. [Abstract] [Full Text] [PDF]
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S. C. Johnson, D. J. Marshall, G. Harms, C. M. Miller, C. B. Sherrill, E. L. Beaty, S. A. Lederer, E. B. Roesch, G. Madsen, G. L. Hoffman, et al. Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet Clin. Chem., November 1, 2004; 50(11): 2019 - 2027. [Abstract] [Full Text] [PDF]
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J. Russell, T. Colpitts, S. Holets-McCormack, T. Spring, and S. Stroupe Defined Protein Conjugates as Signaling Agents in Immunoassays Clin. Chem., October 1, 2004; 50(10): 1921 - 1929. [Abstract] [Full Text] [PDF]
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M. R. Diaz and J. W. Fell High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon J. Clin. Microbiol., August 1, 2004; 42(8): 3696 - 3706. [Abstract] [Full Text] [PDF] |
) and IgG (
) antibodies to 16 different grass allergens.
) or DQA5501+ (•) at 55 °C for 15 min.
(B) Hybridization kinetics. Microspheres coated with
DQA3403- were hybridized to 40 fmol of fluorescent DQA3403+ (
), DQA3401+ (
), or DQA5501+ (•) for 15 min at the
indicated temperatures.