A homogeneous chemiluminescent immunoassay method

Article abstract of DOI:10.1021/ja312039k

A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.

Full text of DOI:10.1021/ja312039k

Communication
pubs.acs.org/JACS
A Homogeneous Chemiluminescent Immunoassay Method
Hashem Akhavan-Tafti,*,† Dean G. Binger,‡,§ John J. Blackwood,‡ Ying Chen,‡ Richard S. Creager,‡
Renuka de Silva,† Robert A. Eickholt,† Jose E. Gaibor,‡ Richard S. Handley,† Kenneth P. Kapsner,‡
Senja K. Lopac,‡ Michael E. Mazelis,† Terri L. McLernon,‡ James D. Mendoza,‡ Bruce H. Odegaard,‡
Sarada G. Reddy,† Michael Salvati,‡,⊥ Barry A. Schoenfelner,† Nir Shapir,‡,¶ Katherine R. Shelly,‡
Jeff C. Todtleben,‡ Guoping Wang,† and Wenhua Xie*,†
†Beckman Coulter Inc., Southfield, Michigan 48033, United States
‡Beckman Coulter Inc., Chaska, Minnesota 55318, United States
S
* Supporting Information
technology utilizes enzyme fragment complementation of β-
ABSTRACT: A new homogeneous chemiluminescent
immunoassay method featuring the use of specific binding
members separately labeled with an acridan-based
chemiluminescent compound and a peroxidase is reported.
Formation of an immunocomplex brings the chemilumi-
nescent compound and the peroxidase into close
proximity. Without any separation steps, a chemilumines-
cent signal is generated upon addition of a trigger solution,
and the intensity is directly correlated to the quantity of
the analyte.
galactosidase to generate a chemiluminescence signal when a
binding event occurs.8 It should be noted here that in the field
of immunoassays, the term “homogeneous” just means that no
separation step is needed; it does not have the same definition
as in physical chemistry, since some homogeneous assay
methods, for instance Ru-based electrochemiluminescence
assays, involve the use of a solid phase. While each of these
technologies has its own uniqueness,9 the quest for new
homogeneous immunoassay technologies continues as both the
clinical diagnostic and life science research communities
constantly look for assay technologies that are sensitive, robust,
and easy to implement.
huge effort has been expended in the field of assay
Adevelopment, particularly immunoassay development, to
simplify the assay process while preserving the essential benefits
in sensitivity, robustness, broad applicability, and suitability to
automation. One approach has been to devise so-called
homogeneous assay formats where no separation of a
detectably labeled specific binding member is needed.1 This
type of methodology relies on devising a detection principle
that is modulated and either turned on or turned off as a result
of the binding reaction. In contrast, heterogeneous assay
formats rely on physical separation of bound and free
detectably labeled specific binding members before quantifica-
tion. Such physical separation, which consists of extensive
washing steps, is not only laborious and time-consuming but
also makes the performance of an assay more complex and
susceptible to cumulative error. A large number of current
homogeneous immunoassays are based on detection of
fluorescence, using fluorescence polarization (FP), fluorescence
resonance energy transfer (FRET), or time-resolved FRET
(TR-FRET).2,3 These assays usually require proprietary or
specialty fluorescent labels and in many cases tend to have
background issues due to autofluorescence of biological
samples and/or interfering compounds.4 Compared with
fluorescence detection, chemiluminescence offers enhance-
ments of 101−102 in sensitivity and a broader dynamic
range.5 Several chemiluminescence-based homogeneous im-
munoassay technologies have recently emerged. These include
singlet oxygen-induced luminescence proximity assays6 and
electrochemiluminescence assays based on the redox reaction
of ruthenium ions proximal to an electrode.7 Another
We recently reported a new class of robust chemiluminescent
compounds for peroxidase detection.10 These acridan-based
compounds, in formulations containing a phenolic enhancer,
generate instantaneous chemiluminescence of superior intensity
when in contact with a peroxidase (Figure 1). Such formulated
solutions have found immediate applications as substrate
reagents for ultrasensitive detection of horseradish peroxidase
(HRP). Recently their superb chemiluminescence intensity as
well as extraordinary stability prompted us to explore the use of
Figure 1. New acridan-based chemiluminescent compounds.
Received: December 10, 2012
© XXXX American Chemical Society
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dx.doi.org/10.1021/ja312039k | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
these compounds as chemiluminescent labels. We report here
our recent study leading to the development of a nonseparation
immunoassay method featuring the use of such a chemilumi-
nescent label and an HRP-conjugated specific binding member
for inducing a chemiluminescent reaction.
established in all of the cases we investigated. More details can
be found in a recently published patent.11
As our investigation continued, the new no-wash assay
method enjoyed success in the case where a capture antibody
and compound 2 were both immobilized on a solid phase,
either a microtiter plate well or a microparticle. However, in the
cases where 2-labeled antibody was used directly in conjunction
with an HRP-conjugated complementary antibody or antigen in
homogeneous assays without any solid phase involved, we saw
less impressive signal-to-noise ratios because of high back-
ground due to the chemiluminescence observed when no target
analyte was present. This remained a challenge until recently,
when another aspect of this no-wash assay method was
discovered.
We recently noted that the performance of no-wash assays
involving the use of solid phases could be further improved by
addition of certain compounds. Among the most effective were
ascorbic acid, 2-aminophenol, hydroxylamines, and certain
other compounds with antioxidant properties. Shown in Table
1 are the results of a no-wash prostate-specific antigen (PSA)
The initial idea was that in an immunoassay using binding
members separately labeled with compound 1 and HRP, the
immunological binding event would bring 1 and HRP into
close proximity, and as a result, the effective concentration of
HRP relative to 1 in the microenvironment of each
immunocomplex would be much higher than in the rest of
the bulk solution, where no binding event would take place. We
reasoned that at given concentrations of both 1 and HRP
during an immunoassay, the formation of a greater number of
immunocomplexes would produce a more intense chemilumi-
nescence signal, even without separation of unbound binding
partners. This would be especially true if the measurements
were to be taken during the initial stage of the chemilumines-
cent reaction, since the chemiluminescence generation is
exceptionally rapid (Figure 1) and, as observed in our previous
experiments, when the concentration of HRP increased
significantly with a given amount of 1, the chemiluminescence
from the system started to change from a glow profile to a flash
profile because of fast depletion of 1.
Table 1. Effect of Selective Signal Inhibiting Agents
no SSIA
Et2NOH added
PSA
signal
reduction
For proof-of-concept experiments, amino-reactive labeling
compounds 2 with the structures shown in Figure 2 were
a
a
signal (ng/mL)
RLU
S/S0
RLU
S/S0
S0
S1
S5
0
15368
−
44
6694
446120
−
67
56%
34%
20%
0.46
129
680002
54298678
3533
43144054
6445
a
Signal intensity measured in relative luminescence units (RLU).
assay using silica microparticles immobilized with both a
monoclonal anti-PSA antibody and compound 2a. Significant
improvements in S/S0 were observed when Et2NOH was added
before signal generation with a trigger solution. While these so-
called selective signal inhibiting agents (SSIAs) caused signal
suppression at all PSA levels, they induced a much more
significant reduction in the background chemiluminescence.
More profoundly, when incorporated into our homogeneous
assays using directly labeled antibodies without any solid phase,
which had previously given less satisfactory results, SSIAs made
such a drastic difference that these assays started to work and
displayed similar or better performance than the corresponding
washed assays. This is considered a significant advancement
because it makes the assay truly homogeneous, a feature desired
for faster immunoreaction kinetics. More importantly, in
comparison with current homogeneous assay technologies,
this new homogeneous immunoassay method has the
advantage that the use of directly labeled antibodies in
conjunction with readily available and widely used HRP
conjugates makes it an easy task to convert any existing
heterogeneous immunoassay to a homogeneous chemilumi-
nescent assay (Figure 3).
Figure 4 shows the results of such a homogeneous
chemiluminescent assay for IL-8, a widely studied cytokine.
Herein, a capture antibody (mouse monoclonal anti-human IL-
8) was labeled with compound 2a following standard
procedures. A solution containing this 2a-labeled capture
antibody and a commercially available HRP-conjugated
detection antibody was incubated with the sample solution in
a microtiter plate well. Without any further steps after the
incubation, a solution of ascorbic acid was added. Lumines-
cence was then generated by injection of the trigger solution
Figure 2. Acridan-labeled chemiluminescent compounds 2.
synthesized and used to label binding members. First, a
microtiter-plate-based mouse immunoglobulin G (IgG) assay
was examined as a quick test. In this case, a white microtiter
plate was coated with 2-labeled sheep anti-mouse IgG antibody.
To the plate were added mouse IgG standard solution and goat
anti-mouse IgG F(ab)2-HRP. Without any washing steps to
remove unbound HRP-conjugated antibodies, a trigger solution
containing hydrogen peroxide and p-hydroxycinnamic acid
(pHCA) was added to generate the chemiluminescence signal.
This signal was measured for 5 s upon addition of the trigger
solution and showed clear dose−response behavior as the
concentration of mouse IgG changed. It was also interesting to
note that in a similar experiment where the plate was coated
with plain unconjugated anti-mouse IgG antibody and then
blocked with 2-labeled bovine serum albumin (BSA) (instead
of native BSA as in a normal blocking procedure), clear dose−
response behavior was once again observed. Here the
chemiluminescent compound 2 was not directly conjugated
to the antibody but sat in close vicinity. This indicated flexibility
in terms of how the labeling could be done.
Encouraged by these initial experiments, we set out to
examine various assay formats in more detail, including
microparticle-based assays. In such cases, the microparticle
carried not only a capture antibody (as in a normal washed
assay) but also compound 2. With a protocol that required no
washing step as described above, calibration curves were easily
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Journal of the American Chemical Society
Communication
Figure 3. A new homogeneous chemiluminescent immunoassay
method.
Figure 5. Results of a homogeneous competitive assay for cAMP.
Assay conditions: 384-well plate; 3 μL of 2b-labeled rat anti-cAMP
antibody (0.275 μg/mL, diluted directly from the labeling reaction
mixture), 3 μL of HRP-cAMP (0.094 μg/mL), and 4 μL of sample
solution; 30 min incubation at room temperature; addition of 1 μL of
2-aminophenol solution (0.69 mM) before injection of 10 μL of
trigger solution and 1 s read.
Figure 4. Results of a homogeneous sandwich immunoassay for IL-8.
Assay conditions: 96-well plate; 20 μL of 2a-labeled mouse anti-human
IL-8 antibody (2.0 μg/mL), 20 μL of HRP-conjugated mouse anti-
human IL-8 antibody (1:500 dilution from a commercial stock
solution), and 30 μL of sample solution; 1 h incubation at room
temperature; addition of 10 μL of ascorbic acid solution (11 mM)
before injection of 100 μL of trigger solution and 2 s read.
value of 2.6 nM, equivalent to 10.4 fmol/well, a sensitivity in
line with or better than those of current methods.4
To evaluate the practical utility of this assay technology, a
method comparison experiment was performed with PSA
against the Beckman Coulter Hybritech PSA assay on a
modified DxI using 69 previously frozen patient samples
(normal and positive). The homogeneous PSA assay with a 4.5
min incubation time achieved a calculated (2 standard
deviations from S0) analytical sensitivity of 0.0065 ng/mL,
similar to that of the Hybritech PSA assay, which includes
washing steps in the protocol. The correlation coefficient with
respect to the Hybritech PSA assay was R = 0.979 with a slope
of 1.021. Figure 6 shows the plot for the clinically significant
range.
and measured on a luminometer. While the simple protocol
consisted of just a one-step incubation followed by injection
and reading, the assay exhibited excellent sensitivity with a limit
of detection as low as 0.64 pg/mL, which is comparable to or
better than those of current washed enzyme-linked immuno-
sorbent assay (ELISA) methods.12
Another surprising yet attractive feature of the homogeneous
assay method is that with SSIAs, one can use an unpurified
antibody directly from the labeling reaction mixture without a
desalting step to remove unconjugated compound 2. This
further simplifies the process of assay development. Addition-
ally, conditions to reduce the assay volume without
compromising the assay performance can be easily worked
out, a benefit when considering reduced reagent consumption
and miniaturization. An IL-8 assay using an unpurified 2a-
labeled antibody and reduced reagent/sample volumes gave
performance similar to that described above.
We performed some studies to investigate the chemical
mechanism of chemiluminescence signal generation during our
assay. Multiple intermediates were identified, indicating the
presence of more than one reactive species during the process
Our homogeneous immunoassay method works equally well
with competitive assays. Figure 5 shows the results of an assay
for cyclic adenosine monophosphate (cAMP) configured using
this method. In this assay, cAMP−HRP competed with cAMP
present in the sample to bind to an anti-cAMP antibody labeled
with compound 2b. The quantity of immunocomplexes
containing both compound 2b and HRP was inversely
correlated to the quantity of cAMP present in the test sample.
Once again, the assay protocol was simple and straightforward,
consisting of a 30 min incubation followed by trigger injection
and reading on a luminometer. Under the conditions indicated
in Figure 5 without optimization, the assay yielded an IC50
Figure 6. Correlation between the homogeneous and Hydritech PSA
assays.
C
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Journal of the American Chemical Society
Communication
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after addition of the trigger solution. These results will be
published separately in due course.
In summary, we have developed a novel homogeneous
chemiluminescent immunoassay method using specific binding
members labeled separately with a chemiluminescent acridan-
based compound and HRP. This new assay methodology
provides a truly homogeneous nonseparation assay technology
with wide applicability, realizing a long-sought goal in the field.
It does not involve complicated reaction schemes or special
constructs. Compared with current homogeneous assay
technologies, the new method is further characterized by its
simplicity in terms of assay format and great ease in
implementing new assays or converting existing heterogeneous
assays.
ASSOCIATED CONTENT
* Supporting Information
Experimental methods and details. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
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P. IVD Technol. 2004, 10, 33. (b) Akhavan-Tafti, H.; de Silva, R. Xie,
W. U.S. Patent 7,247,726, 2007.
AUTHOR INFORMATION
Corresponding Author
hakhavantafti@beckman.com; wxie@beckman.com
■
(11) Akhavan-Tafti, H. U.S. Patent 7,732,153, 2010.
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and sub- to several pg/mL using chemiluminescent methods. Such
information can be found in the manufacturer’s product brochures,
and some is also available from the Internet on the manufacturer’s
Web sites.
Present Addresses
§Syntiron LLC, Saint Paul, MN 55114.
⊥Rapid Diagnostek, Inc., Plymouth, MN 55447.
¶Medgenics Medical Israel, Ltd., Misgav 20179 Israel.
Notes
The authors declare no competing financial interest.
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