ESONN06, Grenoble France

Immunochemistry and biosensors

Danile Altschuh

UMR CNRS/ULP 7175, ESBS. Parc d'Innovation, Boulevard S. Brant

BP 10413, 67412 ILLKIRCH Cedex, France
E-mail: daniele.altschuh@esbs.u-strasbg.fr

Summary

A biosensor is composed of two elements: a biological recognition unit able

to interact specifically with a target, and a transducer able to convert a change
in property of the solution or surface, due to complex formation, into a
recordable signal.

In contrast with conventional bioassays, biosensors allow the detection of

molecular interactions as they take place, without requiring auxiliary
procedures, making them highly attractive for biotechnological applications.

Immunosensors are biosensors in which the recognition element is composed

of antibodies. Antibodies are powerful recognition tools because of their
affinity and specificity of recognition for the target (antigens), and because of
their diversity, potentially allowing the monitoring of any compound.

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 Developing a biosensor

The recognition element (Biology, chemistry)

- target binding specificity and affinity
- stability (regeneration, storage)
- site specific labeling or surface immobilization
-

The transducer (Physics, chemistry)

- signal to noise
- sample consumption
- sample preparation
- quantitative measurement
- valid for all interactions (SPR, fluorescence-based)
-

The instrumentation (Physics)

- Fluidics
- Portability
- Simplicity
- Price
-

1. Introduction to immunology
Non specific and specific immunity
Humoral and cellular responses
The five fundamental features of specific immunity
The three phases of the immune response
Clonal selection

3. Antibodies
Antibody structure
Sequence variability
The Ig domain
Genetic bases of antibody diversity

4. Antigens

5. Antigen-antibody interactions
Antibody production for laboratory use
Binding parameters
Structural studies
Functional studies

6. Bioassays and biosensors

SPR biosensors
Catalytic sensors
Fluorescence molecular sensors
Perspectives

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 Introduction to immunology

Healthy individuals protect themselves against foreign substances (viruses, bacteria) by means
of the different mechanisms of nonspecific (innate) and specific (acquired) immunity.

Nonspecific (innate) immunity is present prior to exposure to foreign substances, does not
discriminate between substances, and is not enhanced by exposure to such substances. It includes
physical barriers (skin, mucous membranes), a series of mechanisms which prevent invasion or
destroy pathogens before infection takes place (low pH of stomach, lysozyme in mucous
secretions, coughing, ciliary movements), and complement activation if the pathogens enter the
blood.

Specific (acquired) immunity is induced or stimulated by exposure to foreign substances, called

antigens (Ag), is specific for each Ag, and increases with each successive exposure to the same
Ag. The specific immune response has two aspects:

- humoral immunity is mediated by antibodies (Ab), which are produced by B

lymphocytes, and is mainly directed against extra-cellular microbes and their toxins.

- cellular immunity is mediated by T lymphocytes, and is mainly directed to

intracellular microbes or substances.

Nonspecific (innate) and specific (acquired) immunity.

Nonspecific response Specific response

First contact with antigen + +

Second contact with antigen + ++++

Non specific Specific
No memory Memory

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Specific immunity: humoral and cellular responses

Humoral response Cellular response

Antigen location Extracellular Intracellular

-Antigen in macrophages
-Antigen in infected cells (virus, parasite)

Responding cell of B lymphocyte T lymphocyte (helper or cytotoxic)

the immune system

Effector mechanism Circulating antibodies -Activation of macrophages (Helper T lymphocyte)

-Lysis of infected cells (Cytotoxic T lymphocyte)

Elimination of the antigen

The five fundamental features of specific immunity :

Specificity

Diversity

Memory

Self-limitation

Ability to discriminate between self and foreign antigens.

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Illustration of specificity

Landsteiner demonstrated the high selectivity of antibodies.

Example: the specificity of antibodies against stereoisomers of tartaric acid.

Forms of tartaric acid used for immunization

COOH COOH COOH

HO H H OH H OH

H OH HO H H OH

COOH COOH COOH

l-tartaric acid d-tartaric acid m-tartaric acid

Reactivity of antibodies when tested against

l-tartaric acid +++ 0

d-tartaric acid 0 +++
m-tartaric acid 0 +++

Illustration of specificity, memory and self-limitation

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

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The three phases of the immune response illustrated for the humoral response

1. Recognition
antigen
B lymphocyte
receptor

2. Activation
Differentiation into
Proliferation and
antibody secreting cell

3. Effector functions

Elimination of antigen
with the help of phagocytes and complement

Specific immunity

The five fundamental features of specific immunity, illustrated for humoral immunity
(antibodies), are specificity, diversity, memory, self-limitation and ability to discriminate
between self and foreign antigens.

The three phases of the specific immune response are

- The recognition phase: the response is initiated by the recognition of foreign Ags by receptors
present at the surface of B or T lymphocytes.

- The activation phase: recognition leads to 1) proliferation of lymphocytes carrying receptors

specific for the Ag and 2) their differentiation from cells able to recognize the Ag into cells
participating into elimination of the Ag. B lymphocytes differentiate into antibody-secreting
cells. T lymphocytes differentiate into cells that activate phagocytes and cells that are able to lyse
antigen-containing cells.

- The effector phase: activated lymphocytes eliminate the Ag. Many effector functions require
the participation of other cells and of the mechanisms of nonspecific immunity.

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Clonal selection

From
Nature 421 , 440 - 444 (23 January 2003)
The double helix and immunology
GUSTAVJ.V.NOSSAL Department of Pathology, The University of Melbourne, Victoria 3010, Australia

Clonal selection

The clonal selection theory (Niels Jerne, David Talmage, Frank Macfarlane
Burnet) implies that the capacity to respond to the huge variety of Ags exists prior
to their introduction into the organism. Clonal selection occurs for B and T
lymphocytes. Today, it can be summarized as follows:

- Every individual contains numerous lymphocytes, each having arisen from a

single precursor cell.

- Each lymphocyte produces only one type of receptor at its surface and is therefore
capable of responding to a distinct epitope (part of the Ag that interacts with the
receptor).

- Antigens select a specific pre-existing clone and activate it, leading to its
proliferation and differentiation.

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Basic antibody structure

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

Basic antibody structure

All antibodies (also called immunoglobulins, Ig) are formed of

two identical light chains and two identical heavy chains, linked
by non covalent bonds and by disulfide bridges.

This basic structure is common to all Ig classes of all vertebrates.

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Amino acid variability in VH and VL

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

Antibody sequences

Ab sequences show different degrees of variability. The light chain can be

divided into two regions, called V (variable) and C (constant).

In heavy chain, 1/4 of the sequence is variable and 3/4 are constant.

Within the variable regions, three regions are hypervariable. They were
called complementarity determining regions (CDR).

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 Light chain folding into two Ig domains

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

Pairwise assembly of Ig domains

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

10
 Antibody chain tracing

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

Location of CDRs on a variable region

Schematic drawing of an IgG Schematic drawing of a Fv

Paratope
VL VL
CL CL
VH VH
CH1 CH1
Fv
VL VH
CH2 CH2

CH3 CH3
Antibody D2.3 (PDB code 1YEC)

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http://rzv054.rz.tu-bs.de/Biotech/
Biotechnology Department Institute for Biochemistry and Biotechnology Technical University Braunschweig
Director Prof. Dr. Stefan Dbel

The Ig domain.

The chains are folded into globular motifs of about 110 amino-acids, formed
of two -pleated sheets.

The light chain contains two such Ig domains (VL: variable light, CL: constant
light).

The heavy chain contains four Ig domains (VH, CH1, CH2, CH3).

The domains are assembled in pairs. The assembly of the variable domain of
the heavy and light chains brings six CDRs into spatial proximity. They form
the paratope or Ab combining site.

The constant regions of antibodies mediate various effector functions (through

their ability ability to bind to cell receptors or other molecules).

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 Ig classes

From
Kuby Immunology 4e
by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunology

http://www.whfreeman.com/immunology/

Isotypes.

In humans, there are five heavy chain isotypes, with different constant
region sequence properties, and called , , , and chains. The five
corresponding classes of Abs have different effector functions and are called
IgG, IgA, IgM, IgD and IgE, respectively.

There are two light chain isotypes, called and .

A given Ab always has two identical light chains and two identical heavy
chains.

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