Fabrication off immunosensor for detection of poultry virus Nghiên cứu chế tạo cảm biến miễn dịch điện hóa để phát hiện virut cúm gia cầm

MINISTRY OF EDUCATION AND TRAINING
HANOI UNOVERSITY OF TECHNOLOGY AND SCIENCE
INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE
---------------------------------------

TRAN QUANG THINH

FABRICATION OF IMMUNOSENSOR
FOR DETECTION OF POULTRY VIRUS
MASTER THESIS OF MATERIALS SCIENCE
Batch ITIMS-2014

SUPERVISOR
Assoc. Prof. Mai Anh Tuan
Dr. Nguyen Hien

Hanoi – 2016


CONTENTS
LIST OF ABBREVIATIONS...................................................................................3
LIST OF TABLES ....................................................................................................4
LIST OF FIGURES ..................................................................................................5
Chapter 1. IMMUNOSENSOR AND IMMUNE REACTION .............................8
1.1. Biosensor and immunosensor ...........................................................................8
1.1.1. Electrochemical immunosensor .................................................................9
1.1.1.1. Transducer ..........................................................................................10
1.1.1.2. Bioreceptor .........................................................................................12
1.1.2. Indirect and direct immunosensor ............................................................12
1.2. Immune Reaction ............................................................................................14
1.2.1. Structure of antibody ................................................................................14

1.2.2. The principle of antibody-antigen interaction ..........................................17
1.2.3. Monoclonal and polyclonal antibody .......................................................23
1.2.4. Immunoglobulin IgG and IgY ..................................................................23
Chapter 2. FABRICATION OF IMMUNOSENSOR .........................................26
2.1. Antibody Immobilization Approaches ...........................................................26
2.1.1. Physical adsorption...................................................................................27
2.1.2. Covalent attachment .................................................................................28
2.1.3. Bio-affinity ...............................................................................................32
2.2. Fabrication of electrochemical sensor based on gold thin film electrodes ....35
2.2.1. Photomask design .....................................................................................35
2.2.2. Main processes in the electrochemical sensor fabrication .......................36
2.2.3. Sensor pretreatment ..................................................................................40
2.3. Antibody Immobilization................................................................................41
2.3.1. Antibody Immobilization using PrA/GA approach .................................42
2.3.2. Antibody Immobilization using SAM/NHS approach .............................43
2.4. Immunoassay Protocol....................................................................................46
Chapter 3. DETECTION OF NEWCASTLE DISEASE VIRUS USING
ELECTROCHEMICAL IMMUNOSENSOR ......................................................47
3.1. Characteristics of electrochemical sensor .......................................................47
3.2. Characteristics of PrA-GA immunosensor .....................................................50
3.2.1. Cyclic voltammetry characterization of PrA-GA immunosensor ............50
3.2.2. Effect of the IgY concentration on the immobilization of PrA-GA
immunosensor ....................................................................................................53
3.3. Characteristics of SAM-NHS immunosensor.................................................54
3.3.1. Cyclic voltammetry characterization of SAM-NHS immunosensor .......55
1


3.3.2. Effect of the pH value on the immobilization of SAM-NHS
immunosensor ....................................................................................................58

3.4. Stability of the signal of ND virus immunosensors ........................................59
3.5. Detection of Newcastle disease virus .............................................................61
3.4.1. Effect of the immunoreaction time ...........................................................62
3.5.2. Sensitivity of Newcastle disease virus immunosensor .............................63
CONCLUSION ........................................................................................................68
REFERENCE ..........................................................................................................69
PUBLICATION.......................................................................................................74

2


LIST OF ABBREVIATIONS
BSA

Bovine serum albumin

CDR

Complementarity-determining region

CE

Counter electrode

CV

Cyclic Voltammetry

DCC

N,N'-Dicyclohexylcarbodiimide

DNA

Deoxyribonucleic acid

EDC

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EID50

50 Percent Embryo Infectious Dose

EIS

Electrochemical Impedance Spectroscopy

GA

Glutaraldehyde

IgG

Immunoglobulin G

IgY

Immunoglobulin Y

LOD

Limit of detection

LOQ

Limit of quantification

ND

Newcastle Disease

NDV

Newcastle Disease virus

NHS

N-hydroxysuccinimide

PCR

Polymerase chain reaction

PrA

Protein A

RE

Reference electrode

TGA

Thioglycolic acid

SAM

Self-assembled monolayer

SD

Standard deviation

WE

Working electrode

3


LIST OF TABLES
Table 1.1 Properties of immunoglobulin classes
Table 2.1. Sputtering parameters
Table 3.1. The crucial parameters obtained from experimental CV data for
fabrication procedures of immunosensor
Table 3.2. Experimental conditions for the attachment of components
Table 3.3. The crucial parameters obtained from experimental CV data for
fabrication procedures of immunosensor
Table 3.4. Experimental conditions for the attachment of components

Table 3.5. The average and standard deviation of Ipeak of sensors
Table 3.6. The crucial parameters obtained from the calibration
Table 3.7. Comparison of analytical properties of different immunosensors for the
detection of Avian Influenza

4


LIST OF FIGURES
Figure 1.1. The performing principle of electrochemical immunosensor
Figure 1.2. Direct and Indirect immunosensor
Figure 1.3. (A) Structure of full-length human anti-PD1 therapeutic IgG4 antibody
pembrolizumab [18], (B) The schematic description of the structure of an IgG
antibody, (C) The domain structure of an IgG antibody
Figure 1.4. X-ray crystallography of the interactions between Fab of 1C1 antibody
and EphA2 antigen
Figure 1.5. Non-covalent bonds in the antigen-antibody interaction
Figure 1.6. The structural difference between IgG and IgY
Figure 2.1. Different orientations of the antibody immobilized on the substrate
Figure 2.3. Pre-treated substrate with maleimide and antibody immobilization by
thiol groups
Figure 2.4. Covalent attachment through carbohydrate residues of antibody
Figure 2.5. Biotinylation of antibody by NHS reagent
Figure 2.6. Avidin-biotin affinity for immobilization
Figure 2.7. Protein A/G-mediated bio-affinity immobilization
Figure 2.8. ssDNA-antibody conjugation to form a hydrazone linker
Figure 2.9. Structure of the integrated electrode
Figure 2.10. Photomask design and detailed structure of electrode sensor
Figure 2.11. Main processes for sensor fabrication
Figure 2.12. Image of electrochemical sensors on a wafer and a complete sensor

Figure 2.13. Electrochemical cleaning and activation of electrodes in sulfuric acid
by CV
Figure 2.14. The schematic description of the fabrication procedures of PrA-GA
immunosensor

5


Figure 2.15. The schematic of antibody immobilization process using SAM-NHS
Figure 3.1. CV curves of sensor with commercial Ag/AgCl RE and Ag/AgCl wire
Figure 3.2. The uniform of sensors
Figure 3.3. The reaction of GA linker with protein A and IgY antibody
Figure 3.4. CV characterization of modified electrode recorded on Au electrode
Figure 3.5. Effect of the antibody concentration
Figure 3.6. The main reactions on the antibody immobilization
Figure 3.7. CV characterization of modification of WE
Figure 3.8. The schematic description of the CV responses of modified electrode
Figure 3.9. Effect of pH value of the immobilization of antibody
Figure 3.10. The average and the SD of Ipeak of the bare Au electrode
Figure 3.11. The schematic description of the ND virus detection mechanism
Figure 3.12. Effect of the immunoreaction time
Figure 3.13. (A) The CV curves of PrA-GA immunosensor (a) in buffer solution
and after assay with (b) 102, (c) 103, (d) 104, (e) 105, (f) 106 EID50/mL ND virus. (B)
the relationship between ΔIpeak and various ND virus concentrations of PrA-GA
immunosensor
Figure 3.14. The relationship between ΔIpeak and various ND virus concentrations

6

INTRODUCTION
Newcastle disease (ND) is one of the most popular infection diseases in
poultry that widely spreads in Southern East Asian countries, including Vietnam. Its
most notable effect is that causes severe economic losses in domestic poultry due to
its highly contagion, especially in chicken. Over the past years, the conventional
qualitative methods (haemagglutination inhibition, agar gel precipitation test and
Latex agglutination test) as well as semi-quantitative analysis (enzyme-linked
immunosorbent assay and immunofluorescence test) were introduced for clinical
diagnosis of ND. Although these methods allow effective determinations ND virus
in infective samples, which require rather complicated procedures for sample
preparations and sophisticated instruments for assays. Thus, it is necessary to
develop methods that offer a simple, rapid, cost-effective analytical strategy, which
can be easily used for applications in contamination studies of ND.
To investigate infection diseases, the fabrication and application of
electrochemical immunosensor have been considerably developed. However, most
of the works have used monoclonal immunoglobulin G (antibody IgG) from
mammalian blood. Egg yolk immunoglobulin (IgY) from chickens can be employed
as an alternate IgG in immunoassay, which offers some advantages with respect to
animal care, high productivity and special suitability in the source of antibodies.
In our work, electrochemical immunosensor using IgY as receptors in
configuration has been developed to detect ND virus. This thesis is organized into
three chapters:
In the first chapter, the basic concepts about immunosensor and fundamental
theory of immune reaction will be introduced.
In the second chapter, the fabrication of electrochemical immunosensor will
be described in detail.
In the last chapter, the characterization of immunosensor carried out with ND
virus will be discussed.

7

Chapter 1

IMMUNOSENSOR AND IMMUNE REACTION
1.1. Biosensor and immunosensor
A biosensor is an analyte device consisting of a biological sensing element
attached a signal transducer, which converts signals of the biological reactions into
measurable

signals

[1].

The

biological

sensing

element

ranges

from

oligonucleotides (DNA or RAN) to enzymes, proteins, cells, antibodies or antigens..
Transducer designed on a solid-state substrate that plays a role converting the
signals recorded from biological sensing element into measurable signals like the
electric signals. Biological reactions are able to lead to that include the changing of

pH value, electronic or ionic transfer, refraction, luminescence, micro mass or
thermal transfer… The biosensors based on antibodies or antigens are known as
immunosensors. Thus, the four most common kind of immunosensors based on the
signal of biological reactions are optical, electrochemical, micro mass and thermal
[2].
North [3] proposed the first concept of the immunosensor in 1985 in which
the bioelement was antibody. Recently, the term immnosensors were described as
the ones that can convert the specific antibody-antigen interactions into measurable
signals. In principle, either antibodies or an antibody-antigen complexes
immobilized on transducer’s surface play the role as a bio-receptor toward a target
element (another antibody or antigen).
Most of the immunosensors are designed that based on the two mechanisms
such as biological catalysis and biological affinity. The biological catalysts are
usually enzymes catalyzing for biochemical reactions, while the biological affinity
bases on the specific interaction of proteins, lectins, receptors, live cells, nucleic
acids, antibodies and antigens [2].

8


The applications of the biosensor and immunosensor comprise a wide range
of tasks, ranging from clinical diagnostics, food safety, industrial processes control,
pollution monitoring, drug discovery, to military and security applications [4]. The
interest in the fields of biosensors is reflected directly in its fast rise in the number
of publications. In 1985, there were approximately 100 papers on this subject and
this number rose to 4500 in 2011. Furthermore, the papers published in 2011 alone
represented more than 10% of all articles ever published concerning the biosensors.
This upward trend can also be seen in the global market for biosensors which
increased from 2 billion US dollars market share in 2000 to 13 billion dollars and
predictions for 2018 show figures around 17 billion dollar mark [5].

1.1.1. Electrochemical immunosensor
According to the IUPAC suggestion of definition for electrochemical
biosensors

[6], an immunosensor is an integrated device consisting of an

immunochemical recognition element in direct spatial contact with a transducer
element. Electrochemical immunosensors employ either antibodies or their
complementary binding partners, i.e. antigens or haptens as biological recognition
elements in combination with electrodes or field-effect transistors. Advantage of
this kind of immunosensor ranges from low sample consumption, reasonable cost of
instrumentations to miniaturization possibility, which are the main reasons for
extensive development of electrochemical immunosensors.

Figure 1.1. The performing principle of electrochemical immunosensor

9


The fundamental performance of electrochemical immunosensor is described as
shown in Fig. 1.1. An electrochemical immunosensor can be classified into three
main components, corresponding to the particular roles in its operating principle,
namely, antibody or antigen as molecular recognizers, electrodes attached
recognizers and performance of a transducer [2].
1.1.1.1. Transducer
Based on the measurement method, the several types of transducer employed
in electrochemical immunosensors field are listed in the following:
+ Potentiometric technique
The fundamental principle of all potentiometric transducers are based on the
Nernst equation

[7] according to which potential changes are logarithmically

proportional to the specific ion activity on the electrodes. The signal is measured as
the potential difference (voltage) between potentiometric transducer electrodes
(working electrodes - WE and counter electrodes - CE). Potentiometric sensors are
used to determine the analytical concentration of some components carrying an
electrical charge in the analyte.
+ Transmembrane potential
This transducer principle is based on the accumulation of a potential across a
sensing membrane. Ion-selective electrodes (ISE) use ion-selective membranes
which generate a charge separation between the sample and the sensor surface.
Similarly, bioreceptors (antigens or antibodies) immobilized on the membrane binds
the corresponding compounds (immune reactions) from the solution at the solidstate surface, which leads to the change the transmembrane potential. The electrode
measuring pH is the most popular ISE.
+ Electrode potential
This transducer is similar to the transmembrane potential sensor. However,
an electrode by itself is the surface for the formation of antigen-antibody

10


complexes, changing the electrode potential in relation to the concentration of the
analyte.
+ Field-effect transistor (FET)
The FET is a semiconductor device used for monitoring of charges at the
surface of an electrode, which have been built up on its metal gate between the socalled source and drain electrodes. The surface potential varies with the analyte
concentration. The integration of an ISE with FET is obtained in the ion-selective
field-effect transistor (ISFET). This technique is also highly potential for the
applications of immunosensors.

+ Amperometric technique
This immunosensors are designed to measure a current flow generated by an
electrochemical reaction at constant voltage. Amperometric immunosensors depend
on the electrical transfer from redox reaction of biocomponents to the surface
electrode. Because most protein analytes are not intrinsically able to act as redox
partners

in

an

electrochemical

reaction,

this

technique

needs

using

electrochemically active labels such as enzyme or redox labels.
+ Conductometric and capacitive technique
These immunosensor transducers measure the alteration of the electrical
conductivity in a solution at constant voltage, caused by biochemical reaction
(enzymatic activities) which specifically generate or consume ions. The capacitance
changes are measured using an electrochemical system, in which the bioreceptor is
immobilized onto a pair of noble metal electrodes (Au or Pt). For immunosensors,

an ion-channel conductance immunosensor mimicking biological sensory function
is used to record effectively the small signaling immune reactions that are usually
lost in the high ionic strength of the solution.

11


1.1.1.2. Bioreceptor
Using antibody or antigen as bioreceptors, also known as immunoassays, is a
striking difference in the fundamental basic of immunosensors comparing with
other biosensors. Generally, conventional electrochemical immunosensors have
antibody molecules immobilized on the surface of electrodes, which recognize
specific antigen in the sample. All types of immunosensor can either operate
through direct or indirect way, which are distinguished through using non-labeled
antibody

or

labeled

antibody,

respectively.

The

direct

electrochemical

immunosensors are able to detect directly the electrochemical changes during the
immune complex formation, while the indirect immunosensors use signalgenerating labels attached on antibodies to detect indirectly antigens.
1.1.2. Indirect and direct immunosensor
Indirect immunosensor
For immnosensors using the labeled antibody, the most frequently used
sandwich-type immunoassay involves a couple of antibodies. As shown in Fig.1.2,
primary antibodies are usually immobilized on an electrode, and sandwiched
immune-complexes are formed among the immobilized antibodies, specific antigens
and labeled antibodies. The detectable signal mainly depends on labeled signal tags,
thus, a great scientific effort has been devoted for developing effective labels.
Enzymes and redox-labels are electrochemical active labels which are used widely
in

indirect

electrochemical

immunosensors,

especially

amperometric

immunosensors [2]. Several enzymes are prominent such as alkaline phosphatase
[8], horseradish peroxidase , β –galactosidase [9], cholinesterase [10] and glucose
oxidase [11], while ferrocene derivatives or In2+ salts [12], redox polymers (e.g.,
polymer [PVP-Os(bipyridyl)2Cl]) [13] are known as notable redox-labels.

12

Figure 1.2. Direct and Indirect immunosensor
Direct immunosensor
Although indirect electrochemical immunosensors are highly sensitive due to
the activation of labels, they have also some weak points. First of all, their
fabrication involves numerous steps, which make experiments more complicated. In
addition, the antigen concentration is not measured in a direct way, but rather based
on the signal generated by label. Therefore, electrochemical immunosensors based
on immunoassays using non-labeled antibodies (label-free antibodies) are
preferably chosen for developments in applications. To work label free is very
attractive, especially for the development of in vitro immunosensors since in allows
real-time measurement without any additional hazardous reagents. In the 1970s,
Janata observed a potential change on an immunosensor that was fabricated by
covering PVC membrane-immobilized Concanavalin-A antibody (ConA) on a
potentiometric electrode [14]. This work is referred the first direct electrochemical
immunosensor that was possible to follow the binding process directly in real time
without any labeling. For the detection of antibodies, in 1984, Keating

[15]

modified an electrode upon a dioxin-ionophore antigen conjugate with a PVC
membrane, which was used in the determination of anti-dioxin antibodies.

13


As the mention above, amperometric immunosensors are considered
unsuitably for the detection directly immune components, which should be
employed with enzyme or redox labels. However, in 2003, the report of Hu [16]
that gold nanoparticles modifying anti-paraoxon antibodies were used on a glassy

carbon electrode to detect directly paraoxon by the cyclic voltammetry
measurement. This detection limit at 12 μg/L was a quite significant result for the
initial amperometric immunosensors without needing any labels.
Over forty years, electrochemical immunosensors followed a direct way have
developed considerably. Most of the studies have focused on the improvement
materials in the components of electrochemical immunosensors such as electrodes,
immobilized substances and electrolytes. Moreover, analytical objects have been
extended progressively, which range from infectious viruses in human or animals,
antigens on cancer cells, pathogens to toxic substances.
1.2. Immune Reaction
1.2.1. Structure of antibody
Antibodies, also known as immunoglobulins (Ig), are glycoprotein molecules
produced by white blood cells in the immune system of vertebrates. They play an
essential role as a critical part of the immune response by specifically recognizing to
particular antigens. An antigen is any harmful substance that causes the production
of an antibody, such as bacteria, fungi, parasites, viruses and chemicals. High
specificity and affinity are the most striking characteristics of the antibody-antigen
interaction. The structural and energetic aspects of antibody-antigen binding are
significant to understand how an antibody specifically recognizes its corresponding
antigen.
Structure of antibody
As shown in Fig. 1.3, the typically structure of an antibody molecule
(immunoglobulin G - IgG) comprises four polypeptide chains linked together by
disulfide bonds (S-S), that divided into two identical light chains (L chains) and two
14


identical heavy chains (H chains). The light chain has two different isotypes, kappa
(κ) chain and lambda (λ) chain, which distributed according to different genes in
chromosomes in mammals. There are five classes of immunoglobulins (IgG, IgA,

IgM, IgD and IgE), which differ in amino acid sequence and number of domains in
the constant regions of the heavy chains (CH). In which, immunoglobulin G (IgG) is
the major type of immunoglobulins (approximately 75% in all of the normal serum),
that is the most extensively investigated.
Table 1.1 Properties of immunoglobulin classes [17]
Properties

IgG

IgM

IgA

IgD

IgE

H-chain type

γ

μ

α

δ

ε

L-chain type

κ or λ

κ or λ

κ or λ

κ or λ

κ or λ

Structure

H2L2

(H2L2)5
+J chain

H2L2
and (H2L2)2
+ SC
+ J chain

H2L2

H2L2

Molecular weight

150×103

950×103

160×103

180×103

190×103

Carbohydrate (%)

3

12

8

10

12

Approximate
concentration in serum
(mg/ml)

10

1

2

0.04

0.0003

H: heavy chain; L: light chain; J: Joining chain; SC: secretory component

In addition, by using the suitable enzymes for hydrolysis of peptide bonds,
an immunoglobulin monomer is cleaved into three fragments. In which, two of the
three fragments are identical which retain the ability to bind antigen, that are called
the Fab fragments (fragments of antigen binding). The third fragment called the Fc
fragment (fragment crystallizable) contains carbohydrate chains, which does not
bind antigen.
The remarkable feature of the antibody molecule is showed by comparison of
amino acid sequences from various immunoglobulin molecules. This shows that
immunoglobulin is composed of various copies of a folding unit of about 110 amino

15


acids, each of which forms an independent similar structure called the
immunoglobulin fold. The N-terminal (NH2 terminal) domain of each polypeptide
(heavy and light chains) is highly variable, while the remaining domains have
constant sequences. These domains are called successively the variable region (V
region) and the constant region (C region). Furthermore, a comparison of V region
sequences shows that variability is not uniformly distributed but concentrated into
three areas called the hypervariable regions.

Figure 1.3. (A) Structure of full-length human anti-PD1 therapeutic IgG4 antibody
pembrolizumab [18], (B) The schematic description of the structure of an IgG

antibody, (C) The domain structure of an IgG antibody
The investigated structures of various antigen-antibody complexes have
demonstrated that the domain structure of the antibody molecule is a β barrel
16


consisting of nine anti-parallel β strands (V regions) and seven C regions, and that
the hypervariable regions are clustered at the end of the variable domain arms, Fig.
1.3C. The antigen-combining site of antibodies is formed almost entirely by six
polypeptide segments, three from light variable domains and three from heavy
variable domains. These segments show variability in sequence as well as in
number of residues which refer single amino acid units making up protein chains.
This variability provides the basis for the diversity found in the binding
characteristics of the different antibodies. These six hypervariable segments are
often called the complementarity-determining regions or CDRs. Thus, the antigenbinding specific is defined by the physical and chemical properties of a binding
surface, which formed by six CDR loops in the antibody. Moreover, other parts of
the V region, exclusively the CDRs, are known as the framework regions.
1.2.2. The principle of antibody-antigen interaction
An understanding of antigen-antibody interactions, particularly those with
protein antigens, plays essential roles for the use of antibodies in clinical diagnosis
and therapy. Antibody-antigen interaction is a chemical one between relevant
antibodies and antigens to form specific antibody-antigen bindings during immune
reaction. Richard J. Goldberg proposed the first correct description of the
interaction in 1952 [19] and it is now known as “Goldberg’s theory” of antibodyantigen reaction. In the typically immune reaction, each antibody binds to several
particular sites of antigen called the antigenic determinants (or epitopes) which
include surface configurations, haptenic groups, specific areas and so on. In
contrast, the molecular determinants within the antibody structure that make
specific interactions with the epitopes are often termed paratope. Antibody
paratopes contain “framework” residues that are amino acid units within protein
chains of CDRs.

For better understanding the principle of the antibody-antigen interaction in
the immunoreaction, it is necessary to base on two following directions: (1)

17


structural features of the binding in the antibody-antigen complex and (2) kinetic of
the antibody-antigen interaction. These directions are also known as static and
dynamic properties in the formation of specific bindings.
The binding in antibody-antigen complex and structure features
Structural studies of specific antibodies and their reactions with antigens
became possible after the development of the techniques of cell hybridization for
the production of monoclonal antibodies of predefined specificity [20]. As the
example in Fig. 1.4, X-ray crystallography appeared as the most effective technique
of choice to determine the precise sites of the molecular interactions of antibodies
with their antigens through crystal structures of complexes between them. The
complex usually is obtained under the crystalline form of the Fab fragments of
antibodies associated with specific antigens. Nowadays hundreds of the threedimensional structures of the antibody-antigen complexes are gained by X-ray
crystallography to provide for the immune epitope database. Furthermore, the
crystal structure determinations of highly specific antibody fragments (Fab)
associated protein antigens show also [21]:
(1) Both the L and H chains of antibodies make extensive contacts with antigens,
although frequently those made by the H chain are more extensive.
(2) The specificity of immuno-reaction is determined by the structure of CDRs on
VH and VL part of antibody; in which, the VH CDR3 encoded by the D (diversity)
segment of genome makes important contributions to binding.
(3) The contacting residues of the antigen are discontinuous in sequence but form a
continuous surface (antigenic determinant or epitope).
(4) The contacting surface of the antibody and antigen often show a high degree of
complementarity.

(5) The contacting surface areas of the antibody-antigen interaction are about 600 to
900 Å2.

18


(6) The formation of multiple bonds by non-covalent interactions as van der Waals
forces, hydrogen bonds, electrostatic forces and hydrophobic forces provides
stability to antibody-antigen complexes.
(7) A large proportion of CDR aromatic residues are appeared in the contacts with
antigen.

Figure 1.4. X-ray crystallography of the interactions between Fab of 1C1 antibody
and EphA2 antigen [22]
(a) Three-dimensional view of the Fab 1C1/EphA2 complex. Fab 1C1 heavy chain and light chain
are shown in magenta and beige, respectively. Human EphA2 ligand binding domain (LBD) is
shown in cyan. (b) Stereographic representations of the intermolecular contacts between human
EphA2 and Fab 1C1 CDRH3. Fab 1C1 and human EphA2 are shown in magenta and cyan,
respectively. Nitrogen and oxygen atoms are shown in blue and red, respectively. The
corresponding interface includes several hydrogen bonds shown as black dotted lines. (c) Fab 1C1
CDRH3 penetrates into a channel of the EphA2 molecule via its predominantly hydrophobic tip.
Sulphur atoms are shown in yellow, whereas the rest of the colour code is identical to that in (b).
(d) The maximum likelihood weighted 2mFo-DFc electron density map is shown around the area
of Fab 1C1 CDRH3 penetration into EphA2. Colour code is identical to that in (b).

In addition, the forces joining the antibody-antigen complex which also
called “weak interaction” are not strong covalent bonds, as shown in Fig 1.5. The
contribution of each of these forces to the overall interaction depends on the
particular antibody and antigen involved. A striking difference between antibody-

19


antigen interactions and most other natural protein-protein interactions is that CDRs
of the antibodies contain many aromatic residues, for examples tyrosine or
tryptophan, in their binding sites. These residues participate manly in van der Waals
and hydrophobic forces, and maybe in hydrogen bonds. These forces operate over
very short ranges and determine the high complementarity of contacting surface. In
contrast, electrostatic forces and hydrogen bonds linking oxygen and/or nitrogen
atoms between charged residues, for example glutamate or lysine, provide specific
features and stability in the antibody-antigen complex. Moreover, other parts of the
V region do not play directly in contact with the antigen but they provide a stable
structural framework for the CDRs and help determine their position and
conformation.

Figure 1.5. Non-covalent bonds in the antigen-antibody interaction
Thus, structural features of the antibody-antigen complexes have enucleated
by X-ray crystallographic analysis, that the hypervariable loops (CDRs) of
immunoglobulin V regions determine the specificity of antibodies. The antibody
molecule contacts the protein antigen over an area of its surface that is
complementary to the surface recognized on the antigen. Electrostatic interactions,

20


hydrogen bonds, van der Waals forces and hydrophobic interactions formed
between residues on the antibodies and antigens all contribute to binding. In which,
the residues in most or all of the CDRs make both the specific and the affinity of the
antibody-antigen interaction.
Kinetic of the antibody-antigen interaction

In the kinetic consideration, if a monovalent antibody fragment is used for
analysis, the equilibrium of antigen-antibody binding is defined as:

At the beginning, a chemical reaction proceeds mostly in one direction, but
the reverse rate progressively increases until the forward and reverse speeds are
equal. At this point, the reaction has reached its equilibrium.
According to the law of mass action at equilibrium, the ratio between the
concentrations of the product ([complex]) and the reactants ([antigen] and
[antibody]) is constant. Keq is called the equilibrium constant and is equal to the
ratio between the association (ka) and the dissociation (kd) rate constant.
[𝑐𝑜𝑚𝑝𝑙𝑒𝑥]
[𝑎𝑛𝑡𝑖𝑏𝑜𝑑𝑦]𝑥[𝑎𝑛𝑡𝑖𝑔𝑒𝑛]

=

𝑘𝑎
𝑘𝑑

= 𝐾𝑒𝑞 = K

(1.2)

In order to improve antigen detection, the ratio between complex and free
antigen should be increased as much as possible. From (1) we have:
[𝑐𝑜𝑚𝑝𝑙𝑒𝑥]
[𝑎𝑛𝑡𝑖𝑔𝑒𝑛]

= 𝐾𝑒𝑞 × [𝑎𝑛𝑡𝑖𝑏𝑜𝑑𝑦]

(1.3)

This can be obtained in two ways: increasing either the equilibrium constant
or the antibody concentration.
It’s known that, the greater the strength of the antibody-antigen binding, the
higher its equilibrium constant is. This relationship is expressed by the Gibbs’
energy (ΔG) of the formation of an antibody-antigen complex, which is given by:

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ln (Keq) = -

𝛥𝐺
𝑅𝑥𝑇

(1.4)

where R is the universal gas constant (8.314472 J·K−1.mol−1) and T is the absolute
temperature (310K at 37 °C).
The free energy of complexes formation represents a balance between
enthalpy (ΔH) and entropy ((ΔS) forces as defined by the equation:

ΔG = ΔH – T x ΔS

(1.5)

In general, antigens and antibodies in solution have to overcome large
entropy barriers before they can form a tight binding. There is a loss of the entropy
of free rotation and translation of the separate molecules as well as a loss of
conformational entropy of mobile segments and of side-chains upon binding. On the

other hand, entropy is gained when water molecules are displaced from the surfaces
that become the new interface, which is in the structures observed by X-ray
analysis. This latter effect is quite significant and it appears that water molecules are
almost totally excluded from the interface by the close contact between antibodies
and antigens. For the non-covalent bonds in the antibody-antigen interaction,
because the formation of hydrogen bonds is exothermal, they are mostly formed by
the enthalpy factor. In contrast, hydrophobic bonds are often driven by the entropy
factor due to their endothermic property.
In the immune technology, it has two terms that are affinity and avidity used
widely to describe strengths of the antibody-antigen binding. As the mention above,
the strength of a single antibody-antigen bond, this obtained from a monovalent
antibody fragment with an epitope, is defined the antibody affinity. However, each
complete monoclonal IgG antibody (or IgY antibody) has two antigen-binding sites
in the Fab fragments and other classes of the monoclonal antibody such as IgM,
IgA, IgD, IgE have more than two antigen-binding sites. Thus, all complete
antibodies are multivalent in their reaction with antigens. Similarly, antigens also
exhibit a multivalent characteristic because they can bind to more than one

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antibody. Avidity is a term that refers to the overall strength of that a multivalent
antibody binds a multivalent antigen.
1.2.3. Monoclonal and polyclonal antibody
Monoclonal and polyclonal IgG antibodies purified from mammalian bloods
are used widely in the immunoassays. Both of them have different advantages that
make them useful for individual applications. Polyclonal antibodies are relatively
quick and inexpensive to produce compare to monoclonal antibodies. However,
monoclonal antibodies are higher specific to detect only one epitope on the antigen
than those of polyclonal antibodies [23]. Traditionally, bigger animals such as

horses, sheep, pigs and rabbits, were used for the production of polyclonal
antibodies, while rats were used as a source of production of monoclonal antibodies.
Nowadays, most frequently chosen mammals for polyclonal and monoclonal
antibody production are rabbits and mice, respectively. A quite significant
disadvantage of the production of antibodies from mammalian bloods is that are the
painful collecting and final sacrificing of animals. Thus, the interest in developing
alternative methods for the traditional production of antibodies is enhanced
increasingly in the sense of animal protection.
1.2.4. Immunoglobulin IgG and IgY
Initially, immunoglobulins that found from avian serum or egg yolk were
classified as IgG-like immunoglobulins. In 1969 Leslie [24] showed experimental
data demonstrating great differences in their structure and proposed the name IgY
(immunoglobulin from egg yolk). Now IgY is recognized as a typical lowmolecular weight antibody of birds, reptiles, amphibian, and as an evolutionary
ancestor of IgG and IgE antibodies that are found in mammals. General structure of
IgY molecule is the same as of IgG with two heavy chains and two light chains. As
shown in Fig. 1.6., the major difference between IgG and IgY is the number of
constant regions in heavy chains (CH), which consists of two structural forms of
IgY: full-length and truncated IgY. The full-length structural IgY found on ducks,

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that has four C regions (CH1 to CH4). The truncated structural IgY found on
chickens, lacking the two terminal domains of the constant region, CH3 and CH4, of
the H chains, thus named as IgY(ΔFc). In addition, both IgY forms are much less
flexible than IgG due to the absence of the hinge between CH1 and CH2, which is a
unique mammalian feature.
The major structure difference between IgG and IgY is reflected in typical
properties of IgY, of which IgY do not bind to protein A or G as well as do not bind
to mammalian antibodies [25].

Figure 1.6. The structural difference between IgG and IgY
Among all sources of avian animals, chicken IgY from egg yolk is most
frequently produced. It provides an excellent substitution for IgG from mammalian
blood in many applications (as a research, diagnostic, detection of antigens…). IgY
was also demonstrated to work in practically all tested immunological methods that
were traditionally developed for IgG, for examples immunofluorescence, immuneenzyme techniques and immunosensors.
In this work, the antibody-antigen interaction between anti-ND virus IgY and
ND virus occurs on a working electrode surface in triple electrode configuration.
This interaction will be investigated using CV measurement that is an important and
widely used electroanalytical technique in many areas of chemistry. From obtained

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