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Articles |
1
National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanjing 210096, Peoples Republic of China.
2
Center for Clinical Laboratory Science, Jiangsu
Province, Nanjing 210009, Peoples Republic of China.
a Author for correspondence. Fax 86-25-7712719; e-mail wqian{at}seu.edu.cn
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: A model polystyrene film was spin-coated directly onto a silicon wafer surface. Atomic force microscopy was used to directly monitor the immobilization of anti-HBs antibodies and their specific molecular interaction with HBsAg. Enzyme immunoassay was also used to characterize functional antibody surfaces.
Results: A mean roughness of 2 Å for areas of 25 µm2 was produced. We found a uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after incubation with HBsAg.
Conclusions: This study confirmed the potential of preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on polystyrene surfaces with controlled roughness. It is expected that such biofunctional surfaces could be of interest for the development of new solid-phase immunoassay techniques and biosensor techniques.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immobilization of biological substances onto polystyrene surfaces is governed by several factors, but currently considerable efforts have been expended primarily in immobilizing biological substances onto microtiter well surfaces with various reagents. Isosaki et al. (15) have reported the successful use of methyl vinyl ether-maleic anhydride copolymer for immobilizing protein ligands. Other methods using pretreated microtiter wells to efficiently immobilize target substances have been reported, e.g., treatment with Triton X-100, poly-L-lysine, or Alcian blue (16)(17)(18) as well as the methyl vinyl ether-maleic anhydride copolymer method. There is no doubt, however, that many facets of ELISA can be improved (19). It is likely that better substrate materials remain to be discovered and that the nature of the interactions between antibody and antigen remains to be explored. We therefore compared polystyrene microtiter wells coated with physically adsorbed antibodies against hepatitis B surface antigen (anti-HBs)1 with silicon wafer substrates coated with site-specifically immobilized anti-HBs antibodies (20)(21). It was interesting to see that the sensitivity of the ELISA could be improved by site-specific immobilization. However, simpler methods for the direct and efficient immobilization of antibodies still remain to be found.
Roberts et al. (22) and Davies et al.
(23) have reported that long parallel ridges exist
on blank polystyrene microtiter well surfaces and that the typical
height from valley to peak is 20–30 nm with a periodicity of 50–250
nm. We also observed such ridges in our experiments using atomic force
microscopy (AFM). The ridges result from the molding process used to
fabricate the microtiter wells. The use of such a polystyrene
microtiter well to immobilize one reactant also means that the eventual
antigen-antibody interaction takes place within a small interface of
unknown dimension and unknown reaction volume. We found that a sandwich
ELISA, in which monoclonal anti-HBs antibodies are immobilized onto
polystyrene microtiter well surfaces, did not always proceed in a
highly sensitive and dose-dependent manner in our experiments. The
amount of hepatitis B surface antigen (HBsAg) bound to immobilized
anti-HBs antibodies showed only a proportional increase with increasing
concentrations of added HBsAg from 1.0 µg/L to 100 µg/L in the dose
curve (0.1–2000 µg/L), and the detection limit was 
1.0
µg/L.
In all future studies, a vital ingredient for ELISA is to understand, control, and optimize the interactions occurring at the two interfaces: (a) between a solid material surface and antibodies, and (b) between immobilized antibodies and their specific antigen molecules. One important task is the preparation of dense, homogeneous, highly specific, highly stable surfaces by immobilizing biomolecules with controlled chemical and topographic properties. It is expected that such biofunctional surfaces would be of high interest for the development of new solid-phase immunoassay techniques and biosensor techniques (24).
We describe here a newly developed method to immobilize antibodies onto ultraflat polystyrene surfaces by physical adsorption. For surfaces in both model studies and applications, the topographies of substrate surfaces are important for immobilized biological substances (25)(26). This is true because biological recognition is characterized by both its (bio)chemical nature and its sophisticated three-dimensional topographic aspects. In our experiments, HBsAg and its monoclonal anti-HBs antibodies were used as a model system. The molecular resolution of AFM images provides an important insight into the exact nature of anti-HBs antibodies after their adsorption and the eventual antigen-antibody interactions. The uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after further incubation with HBsAg was identified. Enzyme immunoassays (EIAs) were also used to detect functional surfaces.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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preparation of ultraflat polystyrene surfaces
Single crystal silicon wafers (n-type) were from the
microelectronics center of Southeast University (Nanjing, China) and
were cleaned with "Piranha solution", a 30:70 mixture (by volume)
of 30 g/L H2O2 and
concentrated H2SO4.
The cleaned wafers were thoroughly rinsed in MilliQ-grade
deionized water, absolute ethanol, and deionized water with
sonication, respectively, and then dried in a stream of
nitrogen. Silicon wafer surfaces were spin-coated with polystyrene
immediately after the cleaning process. A silicon wafer surface was
first wetted with toluene and spun at 2000 rpm for 1 s until the
surface looked dry. As soon as the toluene disappeared from the
surface, spinning was stopped. Polystyrene from microtiter wells was
dissolved in toluene, and 300 µL (10 g/L) was used to coat a silicon
wafer surface for 15 s. Each silicon wafer was then spun to
complete dryness at 2000 rpm (60 s).
immobilization of monoclonal anti-HBs antibodies on
polystyrene surfaces
The silicon wafers spin-coated with polystyrene were mounted on
Teflon cuvettes with a diameter of 7.0 mm. Monoclonal anti-HBs
antibodies (200 µL) at a concentration of 5 mg/L in 50 mmol/L
carbonate buffer, pH 9.6 (coating buffer), were added to each cuvette.
After overnight incubation at 4 °C, the antibody solutions were
removed. The cuvettes were washed twice with deionized water and then
dried under a nitrogen stream for AFM imaging. As a comparison,
antibodies were immobilized on a polystyrene microtiter well surface
using the above experimental procedures.
interactions between yeast HBsAg and immobilized
anti-HBs antibodies
The HBsAg standard at a concentration of 10 µg/L in 50 mmol/L
phosphate-buffered saline (PBS), pH 7.4, was added to each cuvette, and
the cuvettes were incubated for 60 min at 37 °C on a
shaker-incubator. After incubation, the cuvettes were rinsed five times
with 250 µL of Tween 20 (200 mg/L) in PBS and five times with 250
µL of deionized water, and dried under nitrogen for AFM imaging.
afm imaging
Imaging of the surfaces was carried out using a commercial AFM
(Nanoscope IIIa; Digital Instruments). The instrument was operated in
Tapping Mode using silicon cantilevers oscillating with an average
amplitude of 100 nm and a resonance frequency between 200 and
450 kHz. The scanning rate selected was <2 Hz. All of the images
presented here were obtained repeatedly and were stable under the
experimental conditions.
eia detection
A commercial EIA kit for the determination of HBsAg was used in a
standard EIA detection procedure. Silicon wafers (4 x 4 x 1
mm) coated with immobilized monoclonal anti-HBs antibodies were placed
in individual wells of a polystyrene microtiter plate. The blank wells
were preincubated overnight at 4 °C with 10 g/L BSA in PBS to block
nonspecific adsorption of HBsAg or antibodies in the next steps. Each
well was filled with 200 µL of PBS containing 10 g/L BSA for 2 h
at 37 °C and shaken constantly to prevent artifacts produced by the
adsorption of HBsAg or antibodies in the next steps. The BSA solution
was removed, and the wells were rinsed with 250 µL of Tween 20 (200
mg/L) in PBS and 250 µL of distilled water. HBsAg standards (100
µL) at different concentrations in PBS were added to each well for 60
min at room temperature. The HBsAg solution was removed, and wells were
rinsed with 250 µL of Tween 20 (200 mg/L) in PBS and 250 µL of
distilled water. Polyclonal goat IgG labeled with horseradish
peroxidase (HRP) was used as the labeled antibody; 100 µL of HRP-IgG
solution was added to each well. After a 60-min incubation at 37 °C,
the excess HRP-IgG was removed, and each well was washed with 250 µL
of Tween 20 (200 mg/L) in PBS and 250 µL of distilled water. A drop
of solution A (tetramethyl benzidine; 50 µL) and a drop of
solution B (H2O2; 50 µL)
from a standard EIA kit were added to each well. The wells were
incubated for 1 min at 37 °C. Finally, a drop of ending solution (50
µL) was added to each well to stop the reaction. The absorbance at
450 nm (A450) of 150-µL
aliquots from each well was measured by a CliniBIO 128. As a
comparison, the EIA detection procedure for antibodies adsorbed on
polystyrene microtiter well surfaces was the same as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(top panel) shows a representative AFM image of a spin-coated
polystyrene film at a concentration of 10 g/L; the corresponding
roughness data along the cross-section are also indicated (Fig. 1
, bottom panel). The mean roughness (Ra) of the whole image
for areas of 25 µm2 was 2 Å, and the method used to
calculate Ra was the same as described in the literature
(29). This concentration (10 g/L) of polystyrene solution
and the present procedure are appropriate for preparing ultraflat
polystyrene films.
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As a comparison, the AFM image of a standard blank polystyrene
microtiter well surface is shown in Fig. 2
(top panel), and the corresponding roughness data along the
cross-section are also indicated (Fig. 2
, bottom panel). The most
prominent types of structures observed were the long parallel ridges.
The typical height from peak to valley was 20–30 nm with a periodicity
between 50 and 250 nm. The Ra of the whole image for areas
of 25 µm2 was 78 Å, which showed that the surface of a
standard microtiter well is not smooth.
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The topographic images of anti-HBs antibodies physically adsorbed on an
ultraflat polystyrene surface are shown in Fig. 3
. Monoclonal anti-HBs antibodies formed a densely packed
two-dimensional antibody film under our adsorption conditions. The
sample consisted of isolated molecules and packed molecular arrays. The
images of anti-HBs molecules also showed many examples of the
characteristic Y shape (Fig. 3
, arrow).
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The topographic image obtained from anti-HBs antibodies adsorbed on a
standard microtiter well surface is shown Fig. 4
. Anti-HBs antibodies appear to adsorb to the polystyrene
surface in clusters, and although they cover a major part of the well
surface, there are distinct regions where no antibody adsorption has
occurred (Fig. 4
, arrow).
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The AFM topography in Fig. 5
is typical of an adsorbed anti-HBs sample on the ultraflat
surface after incubation with yeast HBsAg solution at a concentration
of 10 µg/L. HBsAg particles captured by adsorbed anti-HBs antibodies
are also indicated (Fig. 5
, arrow). The average diameter of the HBsAg
particles captured by the anti-HBs based on visual inspection was 30
nm.
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In Fig. 6
, the dose curve of an immunoassay on ultraflat polystyrene
surfaces for areas of 16 mm2 is compared with the
immunoassay on standard microtiter wells using the same antibody and
antigen for the same areas. According to the dose curve of the
immunoassay on ultraflat polystyrene surfaces (0.1–2000 µg/L), the
amount of HBsAg bound to the immobilized anti-HBs antibodies increased
proportionally with increasing concentrations of added HBsAg from 1.0
µg/L to 500 µg/L, and then reached a plateau. In the dose curve of
the immunoassay on standard microtiter wells (0.1–2000 µg/L; Fig. 6
), two stages of binding were seen. In the first stage, the amount of
HBsAg bound with coated anti-HBs initially increased with increasing
concentrations of added HBsAg (1–100 µg/L), and then reached a
plateau (100–500 µg/L). In the second stage, a hook effect was
observed when a high concentration of HBsAg was present (500–2000
µg/L). Interestingly, the absorbance values at 450 nm were higher in
the immunoassay on ultraflat polystyrene surfaces. The difference
between immunoassays on two substrates with different topographies
seems to occur at higher concentrations of HBsAg.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10
g/L. The ridges observed on standard blank microtiter well surfaces
result from the molding process used to fabricate the microtiter wells.
From crystallographic data it is known that an IgG antibody
(Mr 146 000) is arranged in three
discrete domains: two Fab fragments and one Fc (30)(31)(32). It
is also known that the hinge region between the two Fab domains is
extremely flexible, and as a consequence, it is usually difficult to
predict the exact conformation, and hence size, of an IgG antibody
adsorbed onto a surface. An estimate of the dimensions that would be
observed by AFM can be made from x-ray crystallographic analysis of
isolated Fab fragments (33) and transmission electron
microscopy images (34)(35), giving an expected
upper limit to the molecular dimensions on the surface of 16–19 nm.
AFM has become an important technique in biology and chemistry because
of its unique ability to image and characterize the structure and/or
function of a biological specimen at the solid-liquid interface. We
have used AFM to directly monitor the adsorption of anti-HBs antibodies
on the ultraflat polystyrene surface. AFM results provided a detailed
understanding of the nature of the interactions between antibodies and
surface. Isolated molecules and packed molecular arrays were observed
in our experiments, and the images of the anti-HBs antibodies also
showed many examples of the characteristic Y shape (Fig. 3
, arrow),
although the adsorption of antibodies on polystyrene can lead to the
loss of protein structure and function. Because of the intrinsic
conformational flexibility of IgG and hydrophobic interactions between
antibodies and surfaces, we do not expect all molecules adsorbed from
solution to exhibit the Y shape. In addition, the image (Fig. 3
)
clearly displayed a densely packed two-dimensional protein film
covering an ultraflat polystyrene surface. The distribution of anti-HBs
antibodies adsorbed on a standard microtiter well surface (Fig. 4
) is
different from that of antibodies immobilized on the above substrate,
and only sparse anti-HBs molecules can be found on a microtiter well
surface. From the above comparison, we know that the topographies of
surfaces are as important for antibody adsorption as other surface
characteristics such as hydrophobicity, surface charge, and
co-adsorption of or exchange with surfactants, copolymers, or
proteins. Perturbation by the surface is known to affect the protein
behavior through both surface chemistry and topography
(25)(26)(36)(37)(38)(39), but the details
remain unknown.
A yeast recombinant, which was constructed by Miyanohara et al.
(40), can produce a HBsAg with properties similar to human
HBsAg. Yeast HBsAg is also immunologically similar to its human
counterpart and is now widely used in vaccines against hepatitis B. The
AFM topography in Fig. 5
is a typical image of anti-HBs antibodies
adsorbed on an ultraflat polystyrene surface after incubation with
yeast HBsAg, and dense HBsAg particles captured by capture anti-HBs
antibodies are also shown (Fig. 5
, arrow). The average diameter of the
HBsAg particles based on visual inspection is 30 nm. The
ultrastructure of yeast HBsAg particles produced by recombinant yeast
cells was examined using high-resolution negative, and ice embedding,
electron microscopy (41). With negative staining, the HBsAg
particles were spherical to slightly ovoid with a mean diameter of 27.5
nm. The size of the HBsAg particles bound by the capture antibodies on
the polystyrene surface measured by AFM was the same as the individual
particle size.
Compared with the immunoassay on standard wells (Fig. 6
), the working
range of the immunoassay on ultraflat surfaces is wider. The difference
between the two immunoassays seems to occur at higher HBsAg
concentrations, which also indicates that increasing the binding
capacity of the capture surface does not improve detection at lower
HBsAg concentrations.
Adsorption of antibodies on surfaces of standard wells is usually too inefficient to give good yields and does not always proceed in a dose-dependent manner. Quantitative data have shown that the method most commonly used to immobilize antibodies in microtiter immunoassays functionally inactivates most of the antibodies (7)(8). Immobilization of antibodies onto ultraflat polystyrene surfaces can improve the performance of immunoassays. We have shown that it is possible to increase the number of active antibody-binding sites by controlling surface roughness.
The demand for increased sensitivity and dose responses in immunoassays has led to the development of new solid-phase materials and new immobilization methods on polystyrene. On the other hand, more work is needed to define the exact nature of the antibodies or antigens that cover the polystyrene surface after their immobilization and the eventual antigen-antibody interactions. It seems appropriate to conclude that in the area of immunoassays now, "the technological cart is ahead of the scientific horse". This study confirmed the potential for preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on a polystyrene surface with controlled roughness. It is expected that such biofunctional surfaces could be of interest in the development of new solid-phase immunoassay and biosensor techniques.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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