This document summarizes a new method called the streptavidin biotinylated protein network (SBPN) method for signal amplification in enzyme-linked immunosorbent assays (ELISAs). The SBPN method utilizes the strong interaction between streptavidin and biotin to form a cross-linked protein network on a surface. This network amplifies detection signals through sequential additions of streptavidin and biotinylated proteins. Experimental results showed the SBPN method achieved a 100-fold lower detection limit compared to commercial amplification methods. The SBPN method provides a low-cost, versatile approach for improving ELISA sensitivity without limitations from pre-formed complexes.

1. ISSN 1359-7345
Chemical Communications
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Wonjae Lee, James R. Carey et al.
Layer by layer assembly of biotinylated protein networks for signal amplification

2. This journal is c The Royal Society of Chemistry 2013 Chem. Commun.
Cite this: DOI: 10.1039/c2cc38233d
Layer by layer assembly of biotinylated protein
networks for signal amplification†
Yu W. Chu,a
Bo Y. Wang,a
Huei-Shian Lin,a
Tai-Yen Lin,a
Yuan-Jen Hung,a
David A. Engebretson,b
Wonjae Lee*c
and James R. Carey*a
Described herein is a unique and inexpensive method that outper-
forms commercial methods that amplify the streptavidin–biotin
recognition event. Amplification induced by streptavidin and
biotinylated protein causes the formation of a large detectable
polymer. This approach enjoys a 100-fold decrease in detection
limit in comparison with the commercial methods.
Enzyme-linked immunosorbent assay (ELISA) is a common
immunoassay that is used to detect a variety of important
human diseases.1
The sandwich ELISA consists of a capture
antibody, an antigen, a detection antibody, and an enzyme that can
react with 3,30
,5,50
-tetramethylbenzidine (TMB). The enzyme
consists of horseradish peroxidase (HRP) that is conjugated to
streptavidin.2
Streptavidin and biotin binding is known to be one of
the strongest interactions in nature (Kd 10À15
M),3
and is utilized to
attach the enzyme to the detection antibody (biotinylated antibody).
The HRP conjugated to streptavidin, called S-HRP, reacts with TMB
to generate a signal that is readily measured using common
laboratory methods.2,4
Unfortunately, the detection limit of ELISA does not readily
permit the detection of low concentrations of bacteria, antibodies,
or rare cancer biomarkers that exist in the blood or other bodily
fluids.5
Since ELISA can be operated within a few hours in the
presence of a high antigen concentration, applying ELISA in
conjunction with a signal amplification step may allow faster
disease diagnosis as compared to the currently used identification
platforms, and permit the detection of disease biomarkers present
in quantities too low to be detected by the standard ELISA.
Studies for improving the diagnostic detection limit of disease
based on the ELISA platform have been carried out for many
years.6
For example, the tyramide signal amplification system
commonly produces 100–1000-fold improvement in sensitivity.7
Common strategies utilize designed signal generating media
(SGM) conjugated to the sandwich complex in ELISA. SGM
discussed in the current literature include but are not limited to
enzymes, metal nanoparticles, quantum dots, fluorescence dyes,
magnetic beads, and organic compounds. SGM produce enhanced
signals such as a magnetic field induced change in resistance,
UV-vis absorbance, fluorescence, and many others.7,8
For example,
in ELISA, the SGM is S-HRP, which oxidizes TMB to give a highly
colored, detectable, and quantifiable color change. By replacing
the S-HRP with other types of SGM in a sandwich-type ELISA
process, the sensitivity, linear dynamic range, and the detection
limit of diagnosis can be improved.
Here, we report a unique signal amplification method that can
be applied to various SGM. The method involves protein poly-
merization (also known as dendritic amplification9
) achieving signal
enhancement by utilizing a streptavidin and biotinylated protein
cross-linking event. This cross-linking event generates a protein
network consisting of streptavidins that are connected to various
biotinylated proteins. The resulting protein network is referred to as
the streptavidin biotinylated protein network, or SBPN.
In the SBPN method, streptavidin is added to the antibody–
antigen complex on a surface, followed by the addition of biotiny-
lated protein. Streptavidin and biotinylated protein are then added
sequentially causing the formation of a protein polymer that is
coupled with the antibody–antigen recognition event on the surface
(Fig. 1). The SBPN method generates a large biotin surface arising
from the biotinylated protein. The protein selected for biotinylation
is bovine serum albumin (BSA) and is biotinylated using the NHS-
PEG4-biotin reagent that is commercially available (see the synthesis
and characterization of BSA-PEG4-biotin, ESI†).10
The optimal sized
surface polymer is produced by performing 20 SBPN cycles (Fig. S12,
ESI†). One layer of streptavidin added to one layer of biotinylated
BSA is defined as one SBPN cycle.
The concept of utilizing strept(avidin) and biotinylated
protein to form a protein complex has been reported and
commercialized as the avidin–biotin–peroxidase complex
(ABC) method.11a
The ABC method is a well-known technique that
stains the antibody–antigen recognition event using the avidin–
biotin interaction,11
and according to Vector laboratories, the
a
Department of Applied Chemistry, National University of Kaohsiung, 700
Kaohsiung University Road, Kaohsiung 811, Taiwan. E-mail: jcarey@nuk.edu.tw;
Fax: +886-7-591-9348; Tel: +886-7-591-9778
b
Department of Chemistry, Oklahoma City University, Oklahoma City, OK, USA
c
College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea.
E-mail: wlee@chosun.ac.kr; Fax: +82-62-222-5414; Tel: +82-62-230-6376
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2cc38233d
Received 15th November 2012,
Accepted 7th January 2013
DOI: 10.1039/c2cc38233d
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Vectastain ABC kit, has been cited over 10000 times.12
The ABC is a
pre-formed complex prepared by mixing a specific ratio of avidin
and biotinylated peroxidase before adding the complex to a
surface.11a
Both the ABC method and the SBPN method utilize
strept(avidin) and biotinylated protein, hence the SBPN method
is somewhat similar to the ABC method. A disadvantage of the
ABC method is that the premixed complex may become too large
and may limit its ability to bind to the biotinylated sandwich
complex due to steric hindrance or size related effects.13
Thus,
the signal of the ABC method may be limited due to the size of
the pre-formed protein complex.
In contrast, the SBPN method is a process that amplifies the
presence of biotin from the initial antibody–antigen recognition
event in a layer by layer fashion. Therefore steric hindrance and size
effects do not preclude amplification as it may in the ABC method.
In addition, after the SBPN cycle is completed, there are numerous
biotins available for the next SBPN layer, whereas in the ABC
method, free strept(avidin) may or may not be available for the next
round of ABC or other SGM. Certainly, in the ABC method there are
no free biotins to permit any available strept(avidin) to bind to the
sandwich complex. Furthermore, the ABC complex is formed by
using numerous HRPs which is an expensive recombinant protein,
whereas the BSA used in the SBPN method is an inexpensive protein.
In order to judge the performance of the SBPN method, we
directly coated biotinylated antibodies on a microtiter plate followed
by blocking with BSA. The plate was coated with antibodies in order
to mimic an antigen–antibody complex used in sandwich ELISA.
S-HRP is the SGM commonly used in an ELISA procedure. In the
following discussion, we refer to the procedure that directly adds
S-HRP to the biotinylated antibody coated microtiter plate as the
direct linked amplification (DLA) method. Here, we used S-HRP as
the SGM for the SBPN method since both the ABC and DLA
methods use HRP as their SGM (see Scheme S1 for the procedural
differences of the ABC, DLA, and SBPN methods (20 cycles), ESI†).
We begin our discussion by highlighting a set of results
(shown in Fig. 2) that are clearly and easily differentiable among
the data collected. The spots are displayed using an artificial
yellow background for easy visualization. Each experimental
condition was performed in three separate wells at the same
time and to verify intraplate data consistency. All experiments
were performed by 3 different technicians on three different
days. Therefore, there are 9 data points in each experiment (see
ESI†), but data from only one technician are shown in Fig. 2 and
3. The blue color is generated from the reaction of HRP and
ready-to-use TMB. Thus, the darker blue spots have more HRP
bound to the surface. The larger amount of HRP produces a
larger signal for detecting the biotinylated antibody on the
microtiter plate surface. From Fig. 2, it is easily seen that the
SBPN method has the strongest signal (darkest blue color).
Indeed, these results also indicate that the SBPN cycles generate
a large biotin surface available for binding to S-HRP.
The color values (average of red, green, and blue values) were
estimated using ImageJ software.14
Using this software, the color
values were selected based on a digital circular zone that was the
size of the well. The smaller the color value measured, the darker
the spot and the stronger the signal. Baseline color values were
measured from a BSA blocked well that contained biotinylated
antibody and TMB. The results of each experiment were subtracted
from the baseline color values to obtain relative color values. The
relative color values represent the change in color values; the larger
the relative color value, the darker the spot.
Fig. 1 Illustration of the SBPN method. (a) The sandwich complex of capture antibody–antigen-detection antibody is formed on a surface. (b) Streptavidin is
introduced to begin the SBPN cycle. (c) Biotinylated protein is added after the streptavidin layer to form the first SBPN cycle. (d) Another layer of streptavidin and
biotinylated protein is added layer by layer to form the second SBPN cycle. (e) There is a large biotin surface covering on the initial antibody–antigen recognition event
when the SBPN method is completed.
Fig. 2 Illustration of the ability to detect surface biotinylated antibodies using
the ABC, DLA, and SBPN methods (20 cycles). Each condition was performed in
three wells at the same time. The darker the spot, the larger the amount of HRP
bound to the surface. The SBPN method (20 cycles) produces the strongest signal
than the other two methods at all concentrations of the biotinylated antibody.
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Fig. 3 summarizes the data shown in Fig. 2 by plotting
relative color values versus surface biotinylated antibody
concentration (see Table S6 for the raw data, ESI†). The plot
reveals that the SBPN method has the strongest signal at the
same concentration of surface biotinylated antibody as the ABC
and DLA methods. Although the SBPN method suffers a higher
background compared to both ABC and DLA methods,
the SBPN method still produces the largest amplification.
Comparing the ABC, DLA, and SBPN methods, the SBPN
method at 30 pg mLÀ1
produces larger relative color values
than those produced by both the ABC and DLA methods at
3000 pg mLÀ1
(see Fig. S14 for a normalized plot, ESI†).
Notably, at 30 pg mLÀ1
of biotinylated antibody on the surface,
the SBPN method (20 cycles) enjoys a 36- and a 112-fold lower
detection limit than the ABC and DLA methods, respectively.
Indeed, 20 SBPN cycles require an additional 1.5 h to perform
as compared to the ABC method. The additional time required
for the SBPN method can be reduced if automation is intro-
duced. We believe that the increase in experiment time is offset
by the signal enhancement and reduction in cost.
To demonstrate that the SBPN amplification is not supplier
dependent, we compared the ABC, DLA, and SBPN methods
using different sources of ABC reagents, streptavidin, and
PBS buffer (see ESI†). The experiments were performed by
4 different technicians on 4 different days. To reduce the
experiment cost, each technician performed only 5 SBPN cycles.
The SBPN method (5 cycles) at all studied concentrations of
biotinylated antibody produced larger relative color values than
both the ABC and DLA methods. These data demonstrate that
the SBPN method is not supplier dependent and that when the
SBPN method is reduced to only 5 cycles, the detection limit
remains lower than both the ABC and DLA methods.
In summary, we have demonstrated a unique protein poly-
merization method that utilizes the biotin–streptavidin recog-
nition event. In order to compare the ABC, DLA and SBPN
methods, we directly coated biotinylated antibodies on a micro-
titer plate surface to function as a mock sandwich assay. By
means of color values, the detection limit of 20 SBPN cycles was
found to be 112- and 36-fold lower than those of DLA and ABC
methods, respectively. Even when the SBPN method is reduced
to only 5 cycles, the detection limit of the SBPN method is still
lower than those of the ABC and DLA methods. The SBPN method
is versatile in that it may be used with any antibody–antigen
combination since initiation of the SBPN method depends
only on the streptavidin–biotin interaction. Finally, the SBPN
method can be applied to other SGM to obtain further
signal improvements. It is important to note that SBPN is
not limited to only immunoassay systems, but can also be
extended to other applications involving the streptavidin–
biotin interaction.
This work was supported by National University of Kaoh-
siung and by the National Science Council (NSC) of Taiwan
under contract numbers NSC 98-2113-M-390-006-MY2 and NSC
100-2113-M-390-001-MY2. The authors thank Prof. Yeung-Haw
Ho and Junho Pak for helpful discussions. The authors also
wish to thank Hao-Ju Chou and Chen-Hao Chen for proof of
concept experiments and useful discussions.
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Fig. 3 Comparison of the ABC, DLA, and SBPN methods (20 cycles). Various
concentrations of biotinylated antibodies are plotted versus the average of
relative color values. Error bars indicate Æ1 standard deviation. Zero pg mLÀ1
represents a control that was performed by the addition of 100 mL per well PBS-1
(see ESI†) to a well that did not contain antibody, but was blocked with BSA.
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