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Journal of Immunological Methods 279 (2003) 33–40
A solid-phase method for evaluation of gold conjugate used in
quantitative detection of antigen by immunogold-labeling
electron microscopy
Ramandeep Kaur1, Manoj Raje*
Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India
Received 14 March 2003; received in revised form 15 May 2003; accepted 10 June 2003
Abstract
Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly
desirable. ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization
of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron
microscopy. The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used
for the final observed and measured signal. In this work, we demonstrate a simple and rapid, solid-phase method in ELISA
format that is also suitable for evaluation and optimization of the gold conjugate. We have demonstrated the utility of this
technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with
immunogold-labeling transmission electron microscopy (TEM) of cell sections. The sensitivity of detection and quantitation of
antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal.
Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this
reagent for immunogold localization and quantification of antigens by TEM.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Gold conjugate; Immunogold labeling; Transmission electron microscopy; Optimization
0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0022-1759(03)00254-0
Abbreviations: TEM, transmission electron microscope; NSB,
nonspecific binding; S/N ratio, signal-to-noise ratio; VHb, Vitre-
oscilla Hemoglobin protein; anti-VHb, polyclonal antiserum against
VHb protein; PBS, phosphate-buffered saline; PBST, PBS contain-
ing 0.01% Tween-20; GP/l2, gold particles/l2.* Corresponding author. Tel.: +91-172-695215; fax: +91-172-
690632/690585.
E-mail address: [email protected] (M. Raje).1 Current address: Department of Biotechnology, Guru Nanak
Dev University, Amritsar-143005, India.
1. Introduction
Immunogold-labeling electron microscopy is a
powerful technique for the localization and quantifi-
cation of antigens in cells and tissues. A crucial
factor for the successful application of this method
is in the ability to decide upon the use of active
reagents at suitable concentrations yielding minimum
nonspecific binding (NSB) while retaining a high
level of signal. As immuno-electron microscopy
experiments are tedious, it is usually a common
practice in most laboratories to carry out some level
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–4034
of prescreening before proceeding to process samples
for the final experiments (Bendayan, 2000). Dot blot
(Brada and Roth, 1984; Moeremans et al., 1984; Van
dePlas, 1997) and Western blot (Paiement and Roy,
1988) prescreening methods have been described
earlier. However, these methods are primarily de-
signed for the qualitative determination of antigen
loss upon exposure to various processing agents rather
than to quantitatively assess the suitability of any
particular reagent for obtaining an optimal signal-to-
noise (S/N) ratio for antigen quantitation by immuno-
gold labeling.
The technical aspects, limitations, and advantages
regarding different types of above assay protocols
have been reviewed earlier (Harlow and Lane, 1988;
Wild, 2001). One of the major drawbacks of mem-
brane-based solid-phase methods is their unsuitability
when minute amounts of reagents are available. Al-
though, to some extent, dot blot assays can be
performed with limited volume of sample, the method
requires relatively larger volumes of reagents. More-
over, the sensitivity of the method is limited, and
therefore, it is more suitable for purified antigens
rather than crude cell or tissue extracts.
Several groups have demonstrated the advantages
of ELISA and related methods for rapid and conve-
nient simulation of processing conditions (fixation,
dehydration, polymerization, etc.), as well as evalua-
tion of reagents for predicting the quantitative detec-
tion of antigen by light and electron microscopy (Craig
and Goodchild, 1982; Leenen et al., 1985; Kaur et al.,
2002a; Ramandeep et al., 2001a). Recently, we have
also demonstrated an effective and simple ELISA-
based method for evaluation and selection of blocking
agents used for immuno-electron microscopy (Kaur et
al., 2002b). This was shown to be superior to other
methods in optimization of signal for detection and
quantitation of antigen by transmission electron mi-
croscopy (TEM). In using ELISA for predicting immu-
nogold-labeling results, one major limitation is that the
protocol involves use of enzyme-labeled secondary
antibodies, which in turn are detected/quantitated by
measuring the intensity of color developed with a
suitable substrate. Evidently, this situation is quite
different from that in case of actual immuno-electron
microscopy of sections wherein the primary antibody
is detected using gold-conjugated molecules. As a
result, the conventional ELISA method cannot provide
any information regarding the reactivity of the gold
conjugate towards primary antibody. In addition, it is
not possible to infer anything regarding the optimum
dilution of gold conjugate for minimizing nonspecific
labeling. Here, utilizing protein A gold conjugate
along with silver enhancement and antigen immobi-
lized in ELISA plates, we demonstrate a solid-phase
assay to cover the above lacunae and provide infor-
mation regarding the gold conjugate to be used for
final signal development.
2. Materials and methods
2.1. VHb antigen and anti-VHb antibody
Escherichia coli DH5a cells harboring the plasmid
PUC8: 15 expressing Vitreoscilla Hemoglobin (VHb)
protein (Dikshit and Webster, 1988) were used as a
source of antigen. Cells from stationary phase were
harvested, washed with phosphate-buffered saline
(PBS), and processed either for electron microscopy
or sonicated in PBS to prepare lysate for the solid-
phase assay. Polyclonal antiserum against VHb pro-
tein was raised in rabbit and used diluted 1:500 with
PBS containing 0.01% Tween-20 (PBST) as described
previously (Ramandeep et al., 2001a).
2.2. Gold conjugate
Colloidal gold (Fisher Scientific) was conjugated
with Protein A (Sigma) using the method described by
Roth (1983). The prepared gold conjugate had an
absorbance maximum at 524 nm (OD= 5.10) and was
shown to bind to rabbit IgG (Bangalore Genie, Ban-
galore, India) as well as the polyclonal antiserum
against VHb protein (anti-VHb) antibody by dot blot
(Van dePlas, 1997).
2.3. Solid-phase assay to determine gold conjugate
reactivity
Evaluation of the optimum dilution of the gold
conjugate was broadly carried out by the method
described previously (Ramandeep et al., 2001a; Kaur
et al., 2002b). However, instead of enzyme-linked
secondary antibody, gold conjugate and silver en-
hancement were used for final signal development.
R. Kaur, M. Raje / Journal of Immunolo
Briefly, into a 96-well ELISA plate, (i) 50 Al of PBSwas added to every first column of eight wells; (ii) 2.5
Ag of cell lysate (in 50 Al PBS) was added to every
second column of eight wells; and (iii) 50 Al of
antibody diluted 1:500 (in PBS) was added into every
third column of eight wells. Coating of the wells was
allowed to take place by incubating the plate over-
night at 4 jC. Subsequently, the wells were blocked
by filling them completely with 3% skim milk (Nestle
Carnation, Glendale, CA) constituted in PBST, for 1
h at room temperature. After three washes with PBST,
50 Al of various dilutions of Protein A gold conjugate
(in PBST) were added to each well and allowed to
react overnight at 4 jC. Subsequently, after washingwith PBST followed by PBS, 50 Al of 2% glutaral-
dehyde (EM Sciences; Ft. Washington, PA) was added
to each well for 30 min. After three washes with PBS
followed by double-distilled water, the bound gold
conjugate was enhanced using 50 Al/well of silver
enhancer reagent (Sigma) for 1 h. The wells were then
washed thoroughly distilled water and OD read at 405
nm in an ELISA reader that had been blanked with a
well exposed to only the silver intensification reagent.
The absorbance of a corresponding strip of eight wells
treated with cell lysate or only PBS was taken to
represent the nonspecific binding by gold conjugate to
cellular components or clear plastic, respectively. The
signal in wells wherein anti-VHb had been coated
represented a positive control for confirmation of the
specific binding to the primary antibody by the gold
conjugate. A comparison between different sets was
done by Wilcoxon’s rank sum test.
2.4. Determination of best blocking agent for gold
conjugate
After determining the optimum dilution of gold
conjugate, the efficiency of various commonly used
blocking agents was evaluated using the same pro-
cedure as described above. Briefly, every alternate
column of eight wells in an ELISA plate were
coated with (i) PBS and (ii) cell lysate. The wells
were then blocked with different blocking agents as
described previously (Kaur et al., 2002a,b). Subse-
quently, after three washes with PBST, 50 Al of goldconjugate (diluted 1:50 in PBST) was added to each
well, and the wells were processed as described
above.
2.5. Processing of cells for TEM
E. coli cells were fixed and processed for embed-
ding in LR White resin and ultrathin sectioning as
described previously (Ramandeep et al., 2001b).
2.6. Immunogold-labeling method
Cell sections on nickel grids were first blocked for
30 min by floating on drops of PBS containing 3%
skim milk dissolved in 0.01% Tween-20 followed by
washing on drops of PBST (5 changes, 5 min each).
To check the level of nonspecific binding by the gold
conjugate, the sections were incubated for 1 h on
different dilutions of protein A gold conjugate. To
evaluate specific binding, a step involving incubation
with rabbit anti-VHb antibody diluted 1: 500 over-
night at 4 jC was incorporated before incubation with
gold conjugate. All antibody dilutions were made in
blocking buffer that had been diluted 10-fold with
PBST. Grids were then washed on drops of PBST and
PBS before being floated on 2% glutaraldehyde for 30
min. Finally, grids were washed on drops of water
before being stained with 2% aqueous uranyl acetate.
2.7. Quantitation of probe density and analysis
The resultant probe density, using test and control
serum, was quantified from random micrographs of
cell sections as described previously (Ramandeep et
al., 2001a). To calculate the probe density, 96 (min-
imum) to 121 (maximum) individual cells were
counted for each sample. At least seven to eight
sections were used for analysis in each case. Labeling
densities were expressed as the number of gold
particles /l2 on cells in section as well as for clear
plastic portions. Comparisons between samples were
made by two-tailed t-test.
gical Methods 279 (2003) 33–40 35
3. Results
3.1. Evaluation of optimum dilution of the gold
conjugate by solid-phase assay
The optimum dilution of protein A gold conjugate
to be used for obtaining minimal levels of nonspecific
labeling (both with clear plastic as well as cellular
Fig. 1. Wells used for the solid-phase assay utilizing gold conjugate
and silver enhancement. (A) Control well, to which PBS was added.
(B) Test well containing 10 ng rabbit IgG. Note the dark intensity at
the base of B as compared to test well A.
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–4036
material) was determined by a solid-phase assay
method. The final signal, in the form of a dark density
at the ELISAwell base (Fig. 1), was obtained by silver
enhancement of gold conjugate bound therein. Using
this method, it was possible to detect as low as 0.6 ng
of rabbit IgG immobilized in the wells (data not
shown). Results of background binding and + ve
signal at various dilutions of the gold conjugate are
presented in Table 1. In uncoated plastic wells, the
absorbance due to nonspecific binding by the gold
conjugate varied 10-fold, i.e., from 0.19F 0.004 to
0.018F 0.006, when the dilution was increased from
1:5 to 1:200. At 1:50 dilution, the absorbance was
0.02F 0.002. Further dilution resulted in almost neg-
ligible decrease in OD. In case of wells coated with
Table 1
Nonspecific and specific binding by different dilutions of gold conjugate
Dilution of
gold conjugate
Nonspecific binding signal of gold conjugate as measu
solid-phase assay and TEM
(OD525 = 5.00) On clear plastic With cellular materia
OD Gp/l2 (TEM) OD Gp/l
1:5 0.19F 0.004 ND 1.21F 0.07 ND
1:10 0.05F 0.003 0.54F 0.068 0.81F 0.02 0.82
1:20 0.03F 0.008 0.32F 0.17 0.31F 0.03 0.36
1:50 0.02F 0.002 0.15F 0.05 0.07F 0.010 0.22
1:100 0.02F 0.003 0.14F 0.07 0.04F 0.004 0.20
1:150 0.019F 0.006 ND 0.04F 0.003 ND
1:200 0.018F 0.006 ND 0.03F 0.003 ND
(1) Clear plastic OD= absorbance in uncoated wellsF S.D. (2) Cellular m
Anti-VHb antibody OD= absorbance in wells coated with 1:500 dilution o
l2F S.E.M. in clear plastic resin portion of sections. (5) Gp/l2 cells = go
indicate number of cells. (6) ND= not done.
cell lysate, the corresponding decrease in absorbance
varied from 1.21F 0.07 to 0.03F 0.003 over the
same dilution range, the OD being 0.07F 0.010 at
1:50 with a near constant value at higher dilutions.
Specific binding of the gold conjugate (primary anti-
body recognition) was confirmed by the signal in
wells coated with anti-VHb antibody. Herein, the
observed OD was 0.85F 0.20 at 1:5 dilution of the
conjugate and 0.19F 0.004 at 1:200 dilution. At 1:50
dilution, the signal (0.30F 0.05) was significantly
higher ( p < 0.001) than that of the uncoated wells or
wells coated with cell lysate. At higher dilutions, a
slight decrease in specific signal was observed.
3.2. Probe density
The number of gold particles/l2 on cell or clear
plastic area for different dilutions of gold conjugate is
given in Table 1. The representative areas are shown
in Fig. 2A–C. On EM sections labeled with 1:10
dilution of protein A gold, the nonspecific signal was
0.54F 0.068 and 0.82F 0.021 gold particles/l2 for
clear plastic and cells, respectively (Fig. 2A).
This decreased to 0.15F 0.05 and 0.22F 0.09 gold
particles/l2 (Gp/l2) ( p < 0.001) at 1:50 dilution of the
conjugate (Fig. 2B). In accordance with solid-phase
assay results, further dilution did not result in any
significant ( p>0.05) decrease in the background sig-
nal (Fig. 2C). When cell sections were probed with
as assayed by solid-phase assay and TEM
red by Positive signal using anti-VHb+ gold conjugate
l/cells Anti-VHb
antibody
Gp/l2
2 (TEM) OD Clear Plastic
(TEM)
Cells (TEM)
0.85F 0.20 ND ND
F 0.021 (121) 0.71F 0.11 ND ND
F 0.11 (116) 0.54F 0.04 ND ND
F 0.09 (116) 0.30F 0.05 0.16F 0.07 3.60F 0.79 (96)
F 0.06 (105) 0.23F 0.02 ND ND
0.20F 0.01 ND ND
0.19F 0.004 ND ND
aterial OD= absorbance in wells coated with cell lysateF S.D. (3)
f anti-VHb antibodyF S.D. (4) Gp/l2 clear plastic = gold particles/
ld particles/l2F S.E.M. on cells in sections, figures in parenthesis
Fig. 2. Electron micrographs of E. coli cells treated with only gold conjugate (A–C) and anti-VHb followed by gold conjugate (D). Dilution of
gold conjugate was 1:10 (A), 1:50 (B and D), and 1:100 (C). The level of nonspecific labeling is highest in (A) and almost the same in (B) and
(C). Specific labeling in (D) is much higher than NSB in (B). Bar = 0.5 Am.
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–40 37
Table 2
Solid-phase assay of nonspecific binding by 1:50 diluted gold
conjugate using various blocking agents
Blocking agent OD plastic OD cell lysate
Nil 0.092F 0.008 0.208F 0.072
3% casein 0.025F 0.10 0.021F 0.005
3% skim milk 0.026F 0.008 0.041F 0.017
10% goat serum 0.048F 0.007 0.106F 0.035
10% horse serum 0.086F 0.008 0.125F 0.013
2% fraction V BSA 0.031F 0.007 0.196F 0.026
2% blot qualified BSA 0.055F 0.007 0.152F 0.027
0.2% fish skin gelatin 0.069F 0.016 0.166F 0.40
0.2% aurion BSA 0.051F 0.018 0.133F 0.042
(1) For details regarding preparation of blocking agents, refer to
Kaur et al. (2002b). (2) OD plastic = absorbance in uncoated wells
F S.D. (3) OD cell lysate = absorbance in wells coated with cell
lysateF S.D.
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–4038
anti-VHb antibody followed by 1:50 dilution of gold
conjugate, the specific labeling density over cells
increased significantly ( p < 0.001) to 3.60F 0.79
Gp/l2, while the nonspecific signal in the clear resin
area remained significantly ( p < 0.001) lower at
0.16F 0.07 Gp/l2 (Fig. 2D).
3.3. Evaluation of blocking efficiency using solid-
phase assay
After determining the optimum dilution of gold
conjugate, the method was used to evaluate the relative
blocking efficiency of various commonly used block-
ing reagents in suppressing nonspecific labeling.
Results of the background binding by gold conjugate
using different blocking agents are presented in Table
2. Solid-phase assay data predicted the lowest levels of
NSB to clear plastic as well as cellular components,
when casein followed by skim milk was used for
blocking. All other blocking agents suggested a sig-
nificantly higher ( p < 0.05) level of nonspecific label-
ing. This is well in agreement with our earlier work
involving TEM of the same samples using gold-
labeled secondary antibodies (Kaur et al., 2002a,b).
4. Discussion
The processing of samples for immunogold label-
ing microscopy is often tedious. Therefore, it would
certainly be worthwhile to have some prior knowledge
regarding the presence of target antigens, as well as
information regarding the quality and optimum con-
centration of reagents to be used to detect them. It is
important that these conclusions be reached by using a
method that mimics the actual immunolabeling proce-
dure as closely as possible and utilizes the same set of
controls and reagents, preferably in as limited quanti-
ties as possible. Any prescreening protocol, however,
suffers from the criticism of not truly representing the
situation in vivo, and the aim should be to use a
method that minimizes this limitation.
Earlier, using cell lysates immobilized in polysty-
rene wells, we have demonstrated the clear advantages
of ELISA-based methods for rapid selection of sample
preparation conditions for quantitative detection of
antigen in cell sections by immunogold-labeling
TEM (Ramandeep et al., 2001a). The immobilized
cell lysate acted as an artificial immunospecimen.
More recently, we have also shown the superiority
of ELISA-based methods for rapid and convenient
simulation of various blocking parameters in the
quantitative detection of antigen (Kaur et al., 2002b)
over other reported methods.
In spite of the clear advantages that ELISA holds
out over other protocols in predicting EM results, one
major drawback has been that the conventional method
utilizes enzyme-labeled secondary antibodies instead
of the gold conjugates, which are used for the final
observed/measured signal in TEM. This therefore does
not truly mimic the situation that exists with samples
used for immunogold labeling. Consequently, besides
deviating from the EM procedure, the method cannot
provide any information regarding the ability of the
gold conjugate to recognize primary antibody or the
optimum dilution to be used for TEM experiments.
Colloidal gold particles, conjugated to antibodies
or other proteins, have proved to be good markers for
the light and electron microscopic detection/quantifi-
cation of antigens in many biological samples (Bees-
ley, 1986; Bendayan, 1985, 1995a,b, 2000; Hainfeld
and Powell, 2000; Horisberger, 1985; Lucocq and
Roth, 1985; Robinson et al., 2000; Roth, 1983). In
addition, independently as well as along with silver
enhancement, they have been widely used for
immuno-detection methods (Brada and Roth, 1984;
Moeremans et al., 1984; Van dePlas, 1997). In the
current study, we have demonstrated a modified
ELISA procedure utilizing gold conjugates, along
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–40 39
with silver enhancement, for final signal development.
This allows the quantitative evaluation of the gold
label regarding optimum dilution and best blocking
agent to be used. The procedure here exploits the fact
that gold probes can be used to bind the primary
antibody, which in turn have been reacted with sample
in an ELISA well. Bound gold is then used to
precipitate silver atoms leading to a measurable in-
crease in optical density. As the primary antibody is
not exposed to prior fixation, the subsequent step of
interaction with gold conjugate is similar to that
encountered in post-embedding labeling, wherein sec-
ondary gold conjugate interacts with primary anti-
body-treated sections on grids.
Our study demonstrated that the modified ELISA
procedure could be easily used to predict the optimum
dilution of gold conjugate to be used for the minimum
level of NSB to clear plastic as well as to other cellular
components. Beyond 1:50 dilution of the conjugate,
the decrease in absorbance due to nonspecific binding
was not significant in both cases. However, the
corresponding specific signal indicating interaction
with the primary antibody remained significantly
higher (OD = 0.30F 0.05, p < 0.001). TEM experi-
ments confirmed this observation, and the dilutions
beyond 1:50 did not result in any further significant
( p>0.50) lowering of NSB. When this method was
used to evaluate the levels of NSB with different
blocking agents, the results gave lowest levels of
nonspecific signal with casein and skim milk. This is
in agreement with our recent work involving quanti-
tative immunogold TEM analysis of NSB as well as
that of other groups who have noted the superiority of
casein and skim milk over other blocking agents in
minimizing nonspecific labeling (Kaur et al., 2002b;
Spinola and Cannon, 1985; Vogt et al., 1987).
Any method useful in providing prior information
regarding the gold conjugate used for TEM should be
able to indicate if that conjugate can (i) recognize
immunoglobulin (bio-activity of the gold conjugate),
(ii) recognize the specific primary antibody used
(specific bio-activity), and (iii) be used to recognize
the antigen being investigated. In all of these above
criteria, the solid-phase assay not only complies but
also provides quantitative information. With rabbit
IgG immobilized in ELISA wells, the protein A gold
conjugate showed positive binding (confirmation of
bio-activity). In addition, it could clearly recognize
immobilized anti-VHb (confirmation of specific bio-
activity). In case of wells coated with antigen con-
taining cell lysate and then sequentially reacted with
primary antibody followed by gold conjugate, the
method provided a clear indication of antigen recog-
nition. Exposure of the cell lysate to fixative as used
for EM, prior to incubation with antibody, did not
result in any significant change in the signal (data not
shown). As silver enhancement is used for final signal
development, the method automatically provides in-
formation regarding reactivity of the enhancement
reagent. This may be important when such steps are
to be included for enhancement of the immunogold
TEM signal. In addition, the ELISA method also
provides information regarding the optimum dilution
of the gold conjugate to be used along with the most
efficient blocking reagent.
The success of any immunodetection experiment
depends, to a major extent, on the reactivity and
quality of the reagents employed, as well as the status
of antigen in sample. Though the most accurate way to
standardize every processing step and reagent is by
trying out every possibility, it is practically not possi-
ble to do so in EM experiments. Consequently, one has
to depend upon some level of prescreening. The final
and one of the vital steps in processing of samples for
immunogold electron microscopy is the visualization
of signal for which gold-conjugated molecules are
used. The solid-phase method described herein would
therefore aid in the rapid selection of a gold conjugate
of desirable activity for successful detection of signal
and also the appropriate dilution to be used for
optimum S/N ratio. By itself, this method cannot
evaluate the effects of sample fixation and other
processing steps on antibody labeling; however, it
should be possible to use this method in conjunction
with the other two ELISA methods described earlier,
i.e., to assess the processing conditions (Ramandeep et
al., 2001a) and also to select the optimal blocking
agent (Kaur et al., 2002a,b) and the decision regarding
the choice of protocols and reagents in immunogold
electron microscopy need not remain empirical.
Acknowledgements
We are grateful to Dr. K.L. Dikshit for providing
the VHb expressing cells and anti-VHb antibody.
R. Kaur, M. Raje / Journal of Immunological Methods 279 (2003) 33–4040
Colloidal gold was the kind gift of Dr. Taposh Dass,
AIIMS, New Delhi. We thank Drs. A. Mondal, G.C.
Varshney, and R. Kishore for critical reading and
correcting the manuscript. Skillful technical assistance
of Mr. Anil Theophilus is gratefully acknowledged.
This is IMTECH Communication No.10/2003.
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