improved fluoroimmunoassays using the dye alexa fluor 647 with the raptor, a fiber optic biosensor

Improved fluoroimmunoassays using the dye Alexa Fluor 647

with the RAPTOR, a fiber optic biosensor

George P. Anderson*, Nandan L. Nerurkar

Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, 4555 Overlook Avenue S.W. Code 6900,

Washington, DC 20375-5438, USA

Received 9 May 2002; received in revised form 18 July 2002; accepted 22 July 2002

Abstract

The performance of the fluorescent dye Alexa Fluor 647 (AF647) was explored as an alternative to Cy5 for immunoassays

on the RAPTOR, a fiber optic biosensor. The RAPTOR performs sandwich fluoroimmunoassays on the surface of small

polystyrene optical waveguides for analyte detection. Fluorescence and immunoassay data were examined at various dye-to-

protein (D/P) ratios for both Cy5 and Alexa Fluor 647. Primarily, due to the self-quenching characteristics of Cy5, Alexa Fluor

647 is substantially more effective in fluoroimmunoassays, yielding over twice the signal for any given analyte concentration.

Alexa Fluor 647 can be attached to antibodies at higher ratios, D/P= 6, before self-quenching begins to limit the dye’s

effectiveness. Furthermore, while Alexa Fluor 647 becomes quenched at high dye-to-protein ratios, D/P= 9, the net

fluorescence yield reaches a maximum, as opposed to Cy5-labeled proteins, which become nearly nonfluorescent at high

labeling ratios, D/Pz 6. The limitations of Cy5 were elucidated with an immunoassay for ricin, while the advantages of Alexa

Fluor 647 were demonstrated in both direct binding assays as well as in a sandwich immunoassay for staphylococcal

enterotoxin B.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Alexa Fluor 647; Cy5; Biosensor; Immunoassay; Fluorophore

1. Introduction

Alexa Fluor 647 (AF647) is a relatively new

fluorescent dye, which has peak excitation at 650

nm and peak emission at 665 nm. It has a molecular

weight of f 1300 and an extinction coefficient of

203,000 cm � 1 M � 1. These properties are similar to

the more commonly employed dye Cy5, whose max-

imum excitation is 649 nm, maximum emission is 670

nm, has a molecular weight of 975, and an extinction

coefficient of 250,000 cm� 1 M � 1. AF647 had been

purported to be more photostable than Cy5 dye

(Panchuk-Voloshina et al., 1999), but has not been

extensively examined for use in fluoroimmunoassays.

While there are a number of fluorescent dyes in the

Alexa Fluor series, only the AF647 was tested, being

the one most compatible for use with the current

excitation/emission requirements (635 nm/>665 nm)

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00327 -7

* Corresponding author. Tel.: +1-202-404-6033; fax: +1-202-

767-9594.

E-mail address: [email protected]

(G.P. Anderson).

www.elsevier.com/locate/jim

Journal of Immunological Methods 271 (2002) 17–24

of the RAPTOR fiber optic biosensor (Fig. 1; Ander-

son and Rowe-Taitt, 2001; Anderson et al., 2000;

King et al., 2000). The RAPTOR is a portable,

automated biosensor that, like its predecessor the

Analyte 2000, is useful for on-site analysis of food,

water, or clinical samples for biological contaminants

(Anderson et al., 1998; Cao et al., 1995; DeMarco et

al., 1999; Ligler et al., 1993; Tempelman et al., 1996).

Thus, we were interested in determining what, if any,

advantage lies in using this new dye to prepare the

fluorescent immuno-reagents for the RAPTOR.

The quenching characteristics of Cy5 at various

dye-to-protein (D/P) ratios have been described

previously by Gruber et al. (2000). This self-

quenching is thought to be caused by formation

of Cy5 dimers; the conjecture being that hydro-

phobic interactions between Cy5 molecules result in

nonrandom binding to the protein surface, and once

immobilized, these deleterious interactions are only

enhanced.

We first set out to confirm those results and to

investigate how using Cy5-labeled antibody at various

D/P ratios impacted the performance of the RAPTOR.

In addition, we wanted to test Cy5-labeled antibody at

its optimal D/P ratio to provide a frame of reference

for judging the effectiveness of AF647. The primary

goal of this study was to examine if another dye,

namely AF647, could significantly increase the over-

all fluorescent signal generated during both direct and

sandwich immunoassays, and thereby improve the

overall limits of detection.

2. Materials and methods

2.1. Buffers and reagents

Ricin was purchased from Sigma (St. Louis, MO).

Antiricin antibodies were provided by Naval Medical

Research Command, Silver Spring, MD. Staphylo-

coccal enterotoxin B (SEB) and affinity-purified

sheep anti-SEB IgG were purchased from Toxin

Technology (Sarasota, FL). RAPTOR wash buffer

consisted of phosphate buffer (Sigma, 8.3 mM, pH

7.3) containing 0.05% (v/v) Triton X-100 (TX-100)

and 0.01% (w/v) sodium azide (PBT). The fluores-

cently labeled antibodies were diluted into a blocking

buffer, which consisted of PBT plus 1 mg/ml casein, 1

mg/ml bovine serum albumin (BSA), and 10 mM

sucrose.

2.2. Waveguide and coupon preparation

Capture antibodies were immobilized onto fiber

optic waveguides by passive adsorption. Injection-

molded, polystyrene fiber optic waveguides (Resear-

ch International, Woodinville, WA) were first black-

ened at their distal ends to prevent reflection of

excitation light. Waveguides were then placed into

capillary tubes (100 Al, cut to 4 cm length) pre-

filled with 38 Al of the appropriate antibody sol-

ution, in general, 100 Ag/ml IgG in 0.1 M sodium

carbonate buffer (pH 9.6). After overnight incuba-

tion at 4 jC, the waveguides were rinsed with

distilled water and glued into the disposable cou-

pons (visible in Fig. 1, lower left). All coupons

were made with three identical waveguides coated

in with captured antibody, and a fourth serving as a

control.

2.3. Preparation of Cy5-labeled rabbit antiricin IgG

Rabbit antiricin IgG was labeled with bisfunctional

NHS-ester Cy5 dye (Amersham, New Jersey). One

vial of Cy5 dye was diluted to 100 Al with water. The

dye was portioned out into 10, 30, and 60 Al aliquots,and each reacted with 300 Ag of antibody in 50 mM

Fig. 1. RAPTOR fiber optic biosensor with optical waveguide

coupon in the foreground.

G.P. Anderson, N.L. Nerurkar / Journal of Immunological Methods 271 (2002) 17–2418

sodium tetraborate, 40 mM NaCl, pH 9.0. The reac-

tions proceeded for 30 min at room temperature in the

dark. Subsequently, labeled protein was separated

from unincorporated dye by size-exclusion chroma-

tography.

2.4. Preparation of AF647-labeled IgG

Alexa Fluor 647 NHS ester (1 mg) was pur-

chased from Molecular Probes (Eugene, OR). The

dye was dissolved in 500 Al of anhydrous acetoni-

trile and divided into 20 Al aliquots, then dried

using an Eppendorf spin vac. These dye aliquots

were sealed under nitrogen and stored at � 20 jCwith desiccant until use.

AF647-conjugated antibodies were prepared at

various D/P ratios. One aliquot of AF647 dye

(f 40 Ag) was first dissolved with 10 Al of DMSO,

then diluted to a final volume of 100 Al with water.

Different volumes of dyes (10, 30, or 60 Al) were

added to 300 Ag IgG (2 mg/ml final concentration) in

50 mM sodium tetraborate, 40 mM NaCl, pH 9.0. The

reaction progressed for 30 min at room temperature in

the dark. Then the labeled protein was separated from

unincorporated dye by size-exclusion chromatogra-

phy. The absorbance at 280 and 650 nm for each

antibody was determined and the D/P ratios were

calculated. This protocol produced goat IgG labeled

at D/P ratios of 2.5, 6.3, and 8.4, respectively. Other

antibodies were labeled with AF647 in a similar

manner; however, the amount of dye required to

obtain similar D/P ratios varied from antibody to

antibody. Most antibodies required f 120 Ag dye/

mg protein to obtain D/P ratios near 6. The fluores-

cence of the labeled antibodies was measured using a

Perkin Elmer LS3 Fluorimeter with a 0.25-cm path-

length cuvette.

2.5. RAPTOR design and operation

Insertion of the assay coupon into the RAPTOR

coupon compartment aligns all necessary optical paths

and engages all fluidic connections required for sam-

ple analysis. A pneumatic pump moves buffer, air,

fluorescent reagent (from on-board reservoirs), or

sample within the system. Serpentine channels in the

coupon provide a common path across the waveguide

surfaces. Bubble detectors, which monitor liquid-to-

air interfaces, control introduction of sample and

fluorescent reagent.

During the two-step sandwich immunoassay, sam-

ple is flowed over the four fiber optic waveguides

mounted in the assay coupon. Antigen present in the

sample binds to the fiber optic waveguide coated with a

corresponding capture antibody; unbound material is

washed away by a brief rinse with PBT. Fluorescently

labeled antibody is next introduced and binds to the

antibody–antigen complexes on the waveguide sur-

face, completing the sandwich assay. This tracer

reagent is maintained at a suitable temperature in an

onboard thermal storage module and recovered after

each assay cycle, allowing multiple sequential analyses

to be performed. Excitation light from four 5 mW

Sanyo laser diodes (635 nm) within the RAPTOR is

focused into the fiber optic waveguides. An evanescent

wave is created along each waveguide, exciting the

fluorescent emission of specifically bound fluoro-

phore-labeled antibodies. The portion of the fluores-

cence captured by the optical waveguide is collimated

by the waveguide’s molded lens and focused onto a

photodiode using a ball lens, chosen for its light-

gathering power and short focal length. A long-pass

dichroic filter (665 nm) rejects reflected laser light.

2.6. Assay procedure

Two RAPTOR assay formats were used, the sand-

wich assay, for which the instrument was designed,

and a simple direct binding assay, where the ligand

itself is fluorescently labeled. The sandwich assay

consists of multiple steps, which are performed auto-

matically by the RAPTOR. Immediately after loading

the coupon into the instrument prior to analyte chal-

lenge, the RAPTOR automatically initiates a 5-min

baseline protocol. The waveguides are briefly rinsed

with PBT (for hydration), and are then incubated for

90 s with fluorescently labeled antibody. Afterwards,

this tracer reagent is returned to its compartment, the

waveguides are rinsed with PBT, and an initial back-

ground wash value is determined. This background

value represents the nonspecific binding of the fluo-

rescent antibody to the waveguide surface and must

be determined before samples are analyzed.

To analyze a sample, it is first loaded into the

sample port using a 1-ml syringe equipped with a

blunt-tipped needle or a transfer pipette. Then the

G.P. Anderson, N.L. Nerurkar / Journal of Immunological Methods 271 (2002) 17–24 19

sample is flowed into the coupon and is incubated

with the waveguides for 7 min. A subsequent wash

with PBT eliminates unbound material; the RAPTOR

automatically flushes the sample port with PBT at the

same time, thus preventing sample carry-over. Next,

the coupon is cleared with air to prevent dilution of

the incoming tracer reagent. Fluorophore-labeled anti-

body reagent is flowed into the coupon and incubated

for 90 s to interrogate the amount of antigen bound to

the waveguides. The tracer reagent is returned to its

reservoir for reuse and a final PBT wash is completed.

The entire standard assay cycle, including all wash

steps, is completed in 10 min.

In direct binding assays, no tracer reagent was used

since the samples contained the fluorescent target,

which was bound directly to the capture antibodies

on the waveguide. For the direct assay, the fluorescent

sample was incubated with the waveguides for the

entire 10 min, with binding being monitored inter-

mittently, and a final reading taken after the wave-

guides were washed.

The rate of fluorescence increase during incubation

with the labeled antibody is automatically calculated

(‘‘assay rate’’), and a final reading is taken to determine

the increase in fluorescence due to antibody bound

during the entire assay cycle (‘‘wash delta’’). Since the

two parameters parallel one another, we only consid-

ered wash deltas for these experiments. Assay data are

stored by the RAPTOR and can be downloaded

through a serial port for quantitative analysis.

2.7. Comparing fluorescent dye capabilities

Comparing Cy5 to AF647 was most efficiently

achieved by testing RAPTOR detection of analyte

concentrations beginning at 1 ng/ml (0.1 ng/ml for

SEB assays). Consecutive assays were then performed

on the same waveguides with increasing concentra-

tions of antigen up to 1000 ng/ml. As a precursor to

the series, all tests began with a blank sample (PBT)

prior to the lowest antigen concentration.

3. Results

The standard protocol for the RATOR was to

prepare Cy5-labeled antibodies at a D/P ratio of 2 to

4. Initial tests examined if signal enhancement could

be achieved by pushing the number of Cy5 dyes per

antibody even higher. Fluorescence measurements

performed for Cy5 incorporated at various ratios onto

rabbit antiricin IgG showed that fluorescence dec-

reased significantly with the addition of Cy5 mole-

cules in excess of the optimal range of 2 to 4 (Fig. 2).

These same rabbit antiricin IgGs, plus an additional

IgG prepared later with a Cy5 D/P ratio of 2.7, were

tested with the RAPTOR to determine the effect of

quenching on the fiber optic immunoassays (Fig. 3).

Not surprisingly, the immunoassay results mirrored

the fluorescence yields. However, despite exhibiting

measurable fluorescence, albeit small, the antibodies

labeled at D/P= 9.2 produced virtually no signal in the

immunoassay. To determine if this poor performance

was due to quenching or loss of activity due to

overlabeling (McCormack et al., 1996), we retested

the same waveguides with the antibody tracer labeled

with a D/P of 1.9. Since these waveguides had been

exposed to 1000 ng/ml of ricin, one would expect an

increase of around 5000 pA had the previous labeled

antibody been inactivated. However, the three wave-

guides in this experiment only had an average increase

of 40 pA, indicating that they had bound the IgG with

the 9.2 D/P and had few additional binding sites for

the D/P 1.9 tracer. Thus, the highly labeled antibodies

were not inhibited from binding to the analyte, but

Fig. 2. Relative fluorescence of Cy5-labeled rabbit antiricin IgG at

constant antibody concentration of 10 Ag/ml (1 ml samples), excited

at 630 nm. D/P: 1.9, solid line; 6.5, dotted line; 9.2, dashed line.


rather, the low fluorescent yield was largely the effect

of self-quenching.

Evidence of Cy5 dimer formation was apparent

from the spectroscopic data. The absorbance spectra

for goat IgG antibodies labeled with Cy5 at three

D/P ratios were compared (Fig. 4). Free Cy5 dye

molecules have an absorption maximum at 650 nm,

with a shoulder at 610 nm. As the antibodies’ D/P

ratio increases, there is a very significant increase

in the relative intensity of the 610 to 650 nm

absorption ratio. This increase in absorbance at

600 nm has been attributed to nonfluorescent dimer

formation (Gruber et al., 2000). The legend for Fig.

4 gives two D/P values for each sample. The first

D/P value given was calculated based only on the

650 nm adsorption. This is followed by an adjusted

dye-to-protein ratio calculated when the anomalous

dye adsorption at 600 nm was also added in as if it

were also 650 nm adsorption.

Fig. 5 shows fluorescence data for goat IgG anti-

bodies at three D/P ratios of AF647 dye. Spectra were

collected similar to the Cy5-labeling data shown in

Fig. 2. Fluorescence increases with the addition of

AF647 molecules to antibody, up to a D/P ratio of 6,

after which fluorescent yield varied little with addi-

tional dye molecules bound to the antibody. The

fluorescence of an antibody labeled with an average

of 3.6 Cy5 molecules each is shown for comparison.

One can note that significant self-quenching of the

Cy5 has already occurred.

To determine the fluorescent yield of AF647 mol-

ecules as the D/P ratio increases, antibodies labeled at

increasing ratios were measured in samples of fixed

dye quantity. This setup permits easy comparison of

their relative quantum yields (Fig. 6), and illustrates

Fig. 4. UV absorbance of Cy5-labeled goat IgG at various ratios,

compared to free dye for reference. D/P: 5, adj 10.5, solid line; 4,

adj 7, dotted line; 3 adj 5.3, dashed line; free Cy5 dye, dashed-doted

line.

Fig. 3. Fluorescent signal increase produced by various ratios of

Cy5-labeled rabbit antiricin IgG on the RAPTOR, upon challenge

with 1 Ag/ml ricin. The average response of three separate

waveguides is plotted.

Fig. 5. Relative fluorescence of AF647-labeled goat IgG antibodies

at multiple dye to protein ratios. Measurements at constant antibody

concentration of 10 Ag/ml, excited at 630 nm. D/P: 8.4, solid line;

6.3, dotted line; 2.5, dashed line. A Cy5-labeled antibody is also

shown, D/P= 3.6, dashed-dotted line.


the amount of self-quenching that the dye exhibits as

more label is added per antibody. Notably, antibodies

labeled at D/P= 2.5 are equal in intensity to free dye

of the same label concentration, meaning virtually no

quenching occurs. As the D/P ratio is increased, the

intensity of fluorescence contributed by each individ-

ual dye molecule decreases. However, as noted in Fig.

5, each AF647 molecule contributes more to the total

fluorescence than it detracts via increased quenching,

thus producing a larger net signal, up to at least a D/P

ratio of 6.

The AF647-labeled antibodies with varying D/P

ratios were tested on RAPTOR both in direct binding

assays and in the more applicable sandwich assays in

order to gauge the performance of AF647 in fluo-

roimmunoassays. The direct assays were useful for

analysis because the surface is already saturated with

binding molecules, and therefore, a rapid and consis-

tently robust fluorescent signal is generated. However,

since the actual test format is the sandwich immuno-

assay, it is vital to ensure that results obtained in the

direct assay correspond to the utilized assay method-

ology. Fig. 7A and B shows the signal generated for

direct binding assays in which samples of 100 and

1000 ng of AF647-labeled goat IgG at various D/P

ratios are bound by rabbit anti-goat-coated optic

waveguides. Similar assays were performed for Cy5-

labeled antibodies, which are optimally labeled (D/

P= 3.6) to provide a direct comparison. Since AF647

has reduced self-quenching, the dye can be added to

antibodies in a higher ratio resulting in fluorescent

signal that peaks in performance at approximately 6

dyes per protein. At this D/P ratio, the signal derived

in the direct binding assay increased an average of

40% over that produced by an optimal amount of Cy5

dye.

A sandwich immunoassay, the standard test for-

mat of the RAPTOR, was performed to confirm

results from the direct binding assays and fluores-

cence data. Sheep anti-SEB IgG labeled at three D/

P ratios with AF647 and at D/P= 3.6 for Cy5, was

used as tracer reagent for the detection of SEB at

various concentrations. Most conclusive is the sig-

nal generated at the two highest concentrations

Fig. 7. Direct binding assays: (A) Wash deltas for AF647-labeled

goat IgG samples (100 ng/ml) at various D/P ratios, black circles.

Cy5 is represented by the open diamond at D/P= 3.6. (B) Same as A

except the goat IgG sample concentration was 1000 ng/ml. Error

bars represent the standard error of the mean of five separate

waveguides.

Fig. 6. Relative fluorescence of AF647-labeled antibodies at

multiple dye-to-protein ratios for constant dye concentration (O.D.

650 nm= 0.1). D/P: 2.5, solid line; 4.0, dashed line; 6.3, narrow

dashed line, and 8.4, dashed-dotted line. Free AF647 dye is also

shown, dotted line.


tested, 10 and 100 ng (Fig. 8). Here, the same

trends observed in the direct binding assays are

repeated, except that for this assay, the antibody

optimally labeled with Cy5 is out-performed by

antibody labeled with AF647 at all tested ratios.

4. Discussion

Fluorimetry data shown in Fig. 5 display the relative

intensity of goat IgG antibodies labeled with AF647 at

various dye-to-protein ratios, as well as a Cy5-labeled

antibody for comparison. Notably, when one attaches

multiple labels to an antibody, the net fluorescence

yield per antibody is much larger for AF647 than for

Cy5. Even at a dye-to-protein ratio of 2.5, AF647

fluoresced more intensely than antibodies labeled with

Cy5 at the higher ratio of 3.6. This difference becomes

even more dramatic if one looks instead at the max-

imum fluorescence yield generated by AF647-labeled

antibodies, i.e. a D/P ratio of 6, which is clearly more

fluorescent than Cy5-labeled antibodies that only

becomes less fluorescent with additional dye mole-

cules, as shown in Fig. 2. Clearly, the improvement

with AF647 is due to reduced self-quenching, since

fluorescence from AF647-labeled proteins approaches

an upper limit, having the same intensity at its optimum

D/P ratio of 6 as at 9 dyes per protein. By comparing

fluorescence yields for samples prepared at a constant

dye concentration (Fig. 6), it is possible to visualize the

net contribution of each dye molecule to the total

fluorescence of the antibody as a whole. Ideally, if no

quenching was encountered, all samples would have

identical curves. However, Fig. 6 illustrates that as the

amount of AF647 dye on a protein is increased, each

dye molecule contributes less to the overall signal

produced by the labeled antibody. At 2.5 dyes per

antibody, the fluorescence is identical to free AF647

dye, where it is assumed there is little to no quenching.

With each additional dye molecule added to the anti-

body, the yield per dye molecule is reduced. Despite

this trend, each additional label still contributes more to

fluorescent signal than it mitigates from the antibody as

a whole, until a threshold of 6 to 7 dyes per protein is

passed, at which point added fluorescence normalizes

with quenching, which results in the upper limit.

Hence, one may conclude that AF647 can be added

to an antibody in greater quantity than Cy5 to increase

fluorescent yield.

RAPTOR translates this fluorescence data into

applicable evidence of AF647’s superiority to Cy5

in fluoroimmunoassays. Juxtaposing first Cy5 and

AF647 in a direct binding assay, such as that

between goat IgG and rabbit anti-goat IgG (Fig.

7), provides a simple comparison of the two dyes.

Comparing the signal increases from Cy5-labeled

goat IgG at a D/P ratio of 3.6, which is in the

optimal range, to AF647-labeled goat IgG at multi-

ple ratios, one can gauge their relative effectiveness.

Using the AF647 dye, the assay signal increased in

direct proportion with the D/P ratio reaching a

maximum at a D/P ratio of 6, consistent with the

fluorescence results (Fig. 5). More importantly, in

direct assays, the AF647 dye (D/P= 6) improved

yields over Cy5 by an average of 50% and 40%,

Fig. 8. Sandwich assays: (A) Wash deltas for varied dye-to-protein

ratios in the detection of 10 ng SEB with AF647-labeled sheep anti-

SEB IgG reagent, black circles. Cy5-labeled sheep anti-SEB IgG is

represented in the above graph by an open diamond at D/P= 2.6. (B)

Results for detection of 100 ng SEB. Error bars represent the

standard error of the mean of at least five separate waveguides.


respectively, at 100 and 1000 ng/ml of goat IgG.

Given that significant variation occurred between

individual assays largely due to waveguide-to-wave-

guide variations, these results were repeated numer-

ous times (n= 6) as were the sandwich immuno-

assay results (n = 7) to improve our confidence in

the data.

After observing promising results for the direct

immunoassay, we examined if AF647 would also

improve the performance of a sandwich immuno-

assay for SEB. Again, the signal is compared at

various ratios of AF647 to antibody and the same

antibody labeled with Cy5 at a D/P ratio of 2.6.

Unlike the direct binding assay, here the average

signal for all the Alexa Fluor-labeled antibodies

was greater than the Cy5-labeled antibody. The

combined average signal increases for the Alexa

Fluor-labeled antibodies at D/P ratios of 4.8 and

7.8, compared to the Cy5-labeled antibody (D/

P= 2.6), were 93% and 136% greater at 10 and 100

ng/ml SEB, respectively. Thus, two different fluo-

roimmunoassays using an AF647-labeled antibody

were shown to give improved fluorescent signals.

5. Conclusion

The focus of this study has been to evaluate the

ability of Alexa Fluor 647 to perform as a fluo-

rescent label for antibodies in sandwich fluoroim-

munoassays for analyte detection in biosensors such

as the RAPTOR. It can be concluded that AF647 is

an effective replacement in assays that have com-

monly used Cy5. Due to the limited quenching

characteristics of AF647, the dye can be added in

greater quantity to an antibody, while still remain-

ing fluorescent. This is obviously a quality critical

to improve detection limits in fluoroimmunosensors.

This reduced quenching also translates into a much

broader range of acceptable D/P ratios. Such a

quality in fluorophores used to label proteins is

invaluable because the labeling process is only

approximate and often can yield unwanted D/P

ratios. Still, if care is taken to achieve optimal D/

P ratios with Alexa Fluor 647, one can significantly

increase the fluorescent signal produced by an

antibody over that achievable with Cy5 in fluo-

roimmunoassays.

Acknowledgements

We thank Lisa Shriver-Lake and Charles Patterson

for their helpful suggestions. This work was funded in

part by the Defense Threat Reduction Agency and by

the U.S. Marine Corps. The views expressed here are

those of the authors and do not represent the opinions

of the U.S. Navy, the U.S Department of Defense, or

the U.S. Government.

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improved fluoroimmunoassays using the dye alexa fluor 647 with the raptor, a fiber optic biosensor

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