AFM study of complement system assembly initiated by antigen-antibody complex
ABSTRACT The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM).
The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab
complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and
30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation
of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information
that can be used to complement other well-established techniques.
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ABSTRACT: Publications in fields of Biosensors, conducting polymers etc.
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ABSTRACT: UV detectors based on surface-barrier structures and new kinds of photoresistive UV detectors have been developed. Their characteristics are presented in comparison with those of other structures.01/2003;
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CEJC 4(1) 2006 194–206
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AFM study of complement system assembly initiated
by antigen-antibody complex
Almira Ramanaviciene1,2, Valentinas Snitka3, Rasa Mieliauskiene1,
Rolandas Kazlauskas1, Arunas Ramanavicius1,2∗
1Department of Analytical and Environmental Chemistry,
Naugarduko 24, 03225 Vilnius 09, Lithuania
2Sector of Immunoanalysis and Informatics, Institute of Immunology,
Moletu pl. 29, 08409 Vilnius 21, Lithuania
3Research Center for Microsystems and Nanotechnology,
Kaunas University of Technology,
Studentu 65, 3031 Kaunas, Lithuania
Received 23 June 2005; accepted 15 November 2005
Abstract: The shape and size of complement system C1 components assembled on a SiO2
surface after classical activation by antigen-antibody complex was determined by tapping mode
atomic force microscopy (AFM). The SiO2substrate was silanized and bovine leukemia virus
proteins gp51 were covalently bound to the SiO2substrate. Self-assembly of complement system
proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was
observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing
solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment
of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active
complement system proteins for 3 minutes and 30 minutes features 2-3 times and 5-8 times
higher in diameter and in height if compared with those observed after formation of Ag-Ab
complex, were observed respectively on the surface of SiO2. This study revealed that AFM might
be applied for the imaging of complement system assembly and provides valuable information
that can be used to complement other well-established techniques.
c ? Central European Science Journals Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.
Keywords: Bionanotechnology, antigen-antibody docking, complement system, bovine leukemia
A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206 195
Biological self-assembling systems are governed by nanoscale processes that evolved over
millions of years, but only the most successful examples of biological systems have been
surveyed. In order to understand the functioning of biological systems, knowledge about
the character of biomolecular interactions and structure of formed complexes is crucial.
The convergence of nano-scale science with modern biology, medicine and bioanalytical
chemistry is a promising trend . Nanotechnology provides the tools and technology
platforms for the investigation and transformation of biological systems, and biology of-
fers inspiration models and bio-assembled components to nanotechnology. Emulating the
concepts and principles of biology has led to the controlled self-assembly of biomateri-
als. Self-assembly and self-organization have inspired ideas for advanced bioengineering
methods at the nano-scale . One of such unique self-assembling bio-nanomachines is a
complement system. The complement system is the major effector of the humoral branch
of the immune system  and plays an essential role in host defense against infectious
agents and in the inflammatory process [4, 5].
The complement system is composed of over twenty proteins involved in a sequence of
cascading reactions ultimately resulting in the elaboration of biologically active products
and destruction of the cellular membranes . The complement system may be initiated
through the “classical pathway” by the binding of antibodies to cell surface antigens,
through the“alternate pathway”by the presence of foreign cell surface components, such
as polysaccharides, as well as antibody aggregates and throught the ”lectin pathway”
by the mannan binding protein [7, 8]. The initial step in the activation of the classical
pathway involves interaction between the first component of complement, C1, and a
number of receptor sites present on the immunoglobulin molecules forming an complexes
. Thus, the most important proteins for self-assembly of complement system are C1
proteins (C1q, C1s and C1r), which are involved in such general complex formation
processes: the complexing of antibody with antigen induces conformational changes in the
Fc portion of the antibody molecule that exposes a binding site for the C1 component of
the complement system; C1 (approximately 900 KDa) exists in serum as a macromolecular
complex consisting of C1q (400 KDa) and two molecules each of C1r (168 KDa) and
C1s (83 KDa), held together in a complex (C1qr2s2) stabilized by Ca2+ions. The C1q
molecule is composed of 18 polypeptide chains that associate to form six collagen-like
triple helical arms, the tips of which bind to exposed C1q-binding sites in the CH2 domain
of the antibody molecule. Each C1r and C1s monomer contains catalytic and interaction
domains; the latter facilitates interaction with C1q or with each other. Each C1 molecule
must bind, via its C1q globular heads, to at least two Fc sites for a stable C1-antibody
interaction to occur .
Following initial activation, various complement components interact, in a highly reg-
196A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206
ulated enzymatic cascade, to generate reaction products that facilitate antigen clearance
and generation of inflammatory response. The complement reaction products amplify the
initial antigen-antibody reaction and convert it into a more effective defense mechanism.
Finally, the terminal components of the complement system generate the membrane-
attack complex . The set of proteins necessary to build a functioning complement
system is present in the blood of all mammalians. However, self-assembling of this sys-
tem starts only in the case if particular antigen-antibody complex is formed on the surface
of cell membrane where antigens are exhibited. Also, circulating Ag-Ab complexes are
able to fix complement system proteins. Due to complement receptors, such immune
complexes are delivered to the organs of the monocyte-phagocytic and reticuloendothe-
lial systems. This process can be affected by environmental contamination . The
complement system of guinea-pig is especially active and is often used as a model system
for investigations of complement system function .
The function of specific actions in complement system is under intensive investiga-
tions: function of complement proteins [14–16]; structure and function of complement
activity controlling protein was predicted ; influence of various factors on regulation of
complement-mediated cytotoxicity ; inactivation of complement system ; etc. Be-
cause of the complexity of the complement system, the real structural model that might be
obtained by AFM will be useful for detailed investigations of this complex system. Appli-
cability of AFM for investigation of other biomacromolecules-based complex systems has
been demonstrated: for structural analysis of the reaction center light-harvesting complex
I , observation of Chitosan-induced restructuration of a mica-supported phospholipid
bilayer , study of single protein based systems , in vitro reconstitution of fibrillar
collagen type I assemblies at reactive polymer surfaces , visualization of band-like cel-
lulose assemblies produced by bacteria , characterization of surface-immobilized layers
of intact liposomes , morphology study of starch, amylose, and amylopectin films 
We believe that atomic force microscopy might be very useful for investigations of
complement system assembly and the aim of this study was to show that complement
system assembly might be investigated by means of tapping mode AFM.
Bovine leukemia virus (BLV) proteins gp51 as well as the specific serum containing anti-
gp51 antibodies were obtained from ‘Biok’ (Kursk, Russia). Ethanol, 3-aminopropyl-
thriethoxysilane (APTES) (Sigma, St.Louis, USA) was used for SiO2substrate prepara-
tion. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimid (EDC) (Sigma, St.Louis, USA) was
used for protein carboxyl group activation. Blood serum of guinea-pig containing active
complement system proteins and BLV not infected cattle blood serum were obtained from
the Institute of Immunology of Vilnius University (Lithuania, Vilnius).
A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206197
2.2Silanization of SiO2substrate
SiO2substrate and test-tubes were washed by ethanol and dried by heating. SiO2sub-
strate was placed into test-tubes with APTES and heated for 1 hour at 90◦C. After
washing with water, SiO2substrate was prepared for protein immobilization.
50 mg/ml solution of gp51 in 0.9 % NaCl was prepared and activated with EDC solution
for 1 hour. Freshly prepared surface of 8˚A SiO2was exposed to activated antigen gp51
molecules for 7 h. Antigen solution used for immobilization gives an approximate area
density of 40-50 molecules per µm2. Active groups that were not engaged in formation
of covalent bonds were deactivated by 0.1 M glycine solution, pH 7.0. The surface was
then rinsed with water.
2.4Preparation of samples for AFM study
Before each AFM measurement the surface of modified 8˚A SiO2was rinsed with water and
dried using a flow of dry Argon gas. To study antigen-antibody interaction, the surface of
8˚A SiO2covered with immobilized antigens was exposed to antibody containing solution
for 30 min., rinsed, dried, and studied by AFM. To study antigen-antibody-complement
system interaction the surface containing antigen-antibody complexes was exposed to
guinea-pig serum rich with active complement system proteins for 1 h and then it was
rinsed, dried and probed by AFM.
Before each AFM-imaging samples were rinsed in 0.1 M glycine buffer, pH 7.0, for 5
min. to prevent non-specific interactions and then thoroughly rinsed in deionized water to
prevent crystallization/aggregation of glycine extent. Then samples were dried by flowing
High resolution tapping mode Atomic force microscope (AFM) measurements were per-
formed with a home-built AFM interfaced with NT-MDT Corp. (Zelenograd, Moscow,
Russia) control electronics and contact mode measurements were made by AFM“Q-Scope
250” Quesant Instrument Corporation (Agoura Hills, USA).
The dry samples were investigated by AFM in the contact and tapping mode in a range
of scan lengths from 5 µm to 1 µm. The commercial Si cantilevers NSG11 series (length
100 µm and width 35 µm) with a force constant 11 Nm−1and tip curvature 10 nm and
resonanse frequency 255 kHz (NT-MDT) were used for tapping mode measurements and
soft cantilevers with force constant 0.25 Nm−1were used for contact mode measurements.
The slides of 0.5 cm2surface area of 8˚A SiO2substrate received from AIXTRON AG
(Aachen, Germany), were used as substrates for AFM investigations.
198 A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206
AFM images are presented in two formats: large format (Fig. 1 A,B,C,D) representing
characteristic image of 5000 × 5000 nm area of modified surface and detailed format (Fig.
3 A,B,C,D) with height width diagrams where characteristic features are analyzed.
For all AFM images 200 × 200 pixels image resolution was applied, scanning rate 10
3 Results and discussion
The immunoreagents bovine leukemia virus (BLV) protein gp51 and polyclonal anti-gp51
were selected for current experiments because of high affinity of these reagents  and
high stability of gp51/anti-gp51 complex [27, 28]. Moreover, BLV infections are still
frequent and detection of those infections as well as effective treatment methods are
important . The experimental concept of the presented experiment was based on the
measurement of radius and height of features observed by scanning probe microscope,
because the height and radius of these features was dependent on the complex size of
immobilized/formed macro molecular species.
AFM imaging was performed in dry state which is a cheaper and more basic alternative
compared to AFM imaging in liquid environment . AFM images of SiO2substrate
surface before and after silanization process seem to have no significant differences in
morphology (Fig. 1A) or any significant change in roughness presented in corresponding
histograms (Fig. 2A), therefore silanization insignificantly changes those properties of
the surface in applied resolution scale. After covalent attachment of gp51 proteins, the
differences in morphology (Fig. 1 B) and roughness (Fig. 2B) of substrate were detectable
at our AFM equipment resolution scale. From the image presented it clearly seems that
180-230 nm diameter and 7-9 nm height (Fig. 3A) features appeared on the surface
of silica. It is estimated that observed features (Fig. 3A) are bovine leukemia virus
protein gp51 and/or its complexes covalently immobilized on the surface of silica substrate
[31, 32]. The results obtained illustrate that success of covalent protein immobilization
can be estimated by AFM.
Optimal incubation time (30 min.) of gp51 modified surface with anti-gp51 Ab was
determined in previous experiments . The AFM images (Fig. 1C, Fig. 3B) registered
after 30 min incubation of gp51 modified silica in solution containing anti-gp51 antibodies
showed significant differences in morphology (Fig. 1C, Fig. 3B) and roughness (Fig. 2C)
if compared with roughness (Fig. 1B, Fig. 3A) and morphology (Fig. 2B) registered
before this treatment or obtained by equally treated control SiO2surface which was not
covered by gp51 (data not shown). In images of mica surface investigated by AFM after
exposure of gp51 modified SiO2to anti-gp51 Ab containing solution (Fig. 1C, Fig. 3B)
two distinct populations of features with height-radius corresponding to those of gp51
and antigen-antibody (gp51/anti-gp51) complex are observed. If gp51 modified surface
was exposed towards solution containing set of other antibodies but not containing any
anti-gp51 observed features (Fig. 3E) were similar to those observed on the gp51 modified
SiO2surface before treatment with antibody containing solution. Expected gp51/anti-
A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206199
gp51 complexes were larger in height-radius almost twice if compared with immobilized
gp51 proteins (Fig. 1B, Fig. 3A). The width-height of gp51/anti-gp51 complex was ap-
proximately twice higher what represents the range of Ag-Ab complex dimensions [34–38].
It allowed to distinguish between immobilized antigens and antigen-antibody complexes
formed. The formed features allow us to conclude that we observe the antigen-antibody
docking reaction, or the appropriate lack thereof, between single antigen molecules im-
mobilized on the surface and single or multiple antibody molecules. However, the yield
of such formations (surface area covered by 7-9 nm height features as presented in fig-
ure 2A was compared with area covered by 10-20 nm height features was measured and
compared) not exceeded 7-15 % if compared with suspected gp51 immobilization yield,
the main reason for it might be improper orientation of epitopes present in gp51, and
this is in good agreement with the results obtained during the AFM detection of Ag-Ab
complexes . Since a high number (over 85 %) of immobilized antigen was not engaged
in the formation of antigen-antibody complex we conclude that not all antigen molecules
are properly oriented and/or denatured and they are unable to bind with Fab sites of
When a surface without immobilized antigens was exposed to the same antibody
containing solution no clear features similar to those presented in Figures 1C and 2B
were detected. The AFM image was absolutely identical to the image of unmodified SiO2
substrate surface because in the absence of immobilized antigen specific affinity interaction
and strong binding of any proteins to the surface was not reliable, not specifically adsorbed
proteins were removed by 0.1 M glycine buffer, pH 7.0.
After 3 min. treatment of silica substrate modified with gp51/anti-gp51 Ab com-
plex in the complement protein containing solution, 40-70 nm diameter and 30-40 nm
height structures appeared on the surface (Fig. 1D and Fig. 3C). Height histogram (Fig.
2D) derived from figure 1D shows appearance of higher features if compared with those
presented in figures 2A,B,C derived from figures 1A,B,C correspondingly. No similar
structures were observed if unmodified silica substrate or silica modified just with gp51
was treated by the same complement system protein containing solution for the same
A significant increase in geometrical dimensions of the detected structure of previously
formed Ag-Ab complex might be the result of self-assembly of a number of C1 components
during early stage of complement system self assembly initiated by antigen antibody
After 1 h treatment of silica modified with gp51/anti-gp51 Ab complex in the com-
plement protein containing solution, sparse 500 nm diameter and almost 400 nm height
structures appeared on the surface (Fig. 1E and Fig. 3D). Appearance of significantly
higher features was confirmed by appearance of significantly higher structures in cor-
responding histogram (Fig. 2E). We believe that those structures were based on com-
plement system proteins self-assembled on the basis of Ag-Ab complexes, while no such
structures were detected if just gp51 modified silica substrate or unmodified silica surface
was treated by the same complement system protein containing solution for the same
200 A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206
After exposure of antigen-antibody complex containing surfaces to complement sys-
tem containing solution we observed features larger in lateral dimension as well as in
height than either gp51 or antigen-antibody complexes. The image appears to show com-
plex formation, what was expected. The area density of the features is about 5-7 times
smaller if compared with the density of antigen-antibody complexes, and about 20-25
times smaller if compared with the density of immobilized gp51; it allows us to suppose
that some spatial factors are crucial for the formation of antigen-antibody-complement
complexes. We favor the notion that the relatively large antigen-antibody complexes may
exist in a wide range of orientations and conformations all of which are not optimal for
antigen-antibody-complement complex formation. Moreover, since the mixture of anti-
gp51 antibodies is polyclonal, several antibodies can bind respectively to different epitopes
of gp51 and such complex might be inconvenient for the formation of antigen-antibody-
complement components complex. Tapping mode phase images (Fig. 1E) clearly show
at least two distinct populations of features with different rigidity that are forming com-
plement system protein complex/complexes. This fact illustrates that different proteins
in the complement system posses different rigidity and might be distinguishable based on
this property by AFM phase imaging.
4 Conclusions and future developments
Our experiments demonstrate that AFM is usable for observing biological molecular pro-
cesses like antigen-antibody docking and even for the formation of larger biological struc-
tures. However, from the AFM data presented in this study it is still not possible to clearly
identify separate proteins, since the detection was performed in at least a partially dried
state and the protein complex appeared in an almost globular shape.
The results presented encourage a detailed AFM study of complement assembly by
application of purified complement system proteins.
This work was financially supported by Lithuanian State Science and Studies Foundation
project number C 03047, and COST action D33.
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204 A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206
Fig. 1 Tapping mode AFM images of (A) silanized SiO2and the same surface consecu-
tively treated with: (B) gp51 for 2 h; (C) anti-gp51 for 30 min; (D) active complement
system proteins for 3 min; (E) active complement system proteins for 60 min. Images
covers area of 5000 × 5000 nm.
A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206205
Fig. 2 Frequency diagrams of tapping mode of corresponding AFM images presented in
Fig. 1. of (A) silanized SiO2and the same surface consecutively treated with: (B) gp51
for 2 h; (C) anti-gp51 for 30 min; (D) active complement system proteins for 3 min; (E)
active complement system proteins for 60 min. Images covers area of 5000 × 5000 nm.
206 A. Ramanaviciene et al. / Central European Journal of Chemistry 4(1) 2006 194–206
A) B) C)
Fig. 3 Height-width study of typical features observed by tapping Mode AFM image of
silanized SiO2surface consecutively treated with: (A) gp51 for 2 h; (B) anti-gp51 for 30
min; (C) active complement system proteins for 3 min; (D) active complement system
proteins for 60 min. Images cover area of 1000 × 1000 nm. Height profiles are shown in
the lower panels for the black lines in the corresponding image; (E) control experiment
silanized SiO2treated by gp51 for 2 h and BLV not infected bovine blood serum (not
containing anti-gp51) for 30 min.