www.elsevier.com/locate/jim

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: manoj@imtech.res.in (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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