PTT 202

Organic Chemistry for Biotechnology

Lecture 7:
Immunological Methods
Zulkarnain Mohamed Idris
zulkarnainidris@unimap.edu.my

Semester 1 2014/2015

Introduction

Antibody: protein produced by animal as a result of the

introduction of foreign substance into its tissue (in process
known as the immune system).
Antigen: foreign substance/molecule or immunogen that
reacts with a antibody.
Hapten: small substance that is not capable to initiate an
immune response but may do so when attached to a larger
molecule.
Epitope: part of antigen that interacts with an antibody.
Paratope: the complementary portion of the antibody that
interacts with an epitope.
Antigen-antibody reactions provide the basis of useful
methods of qualitative and quantitative (immunoprecipitation,
radioimmunoassay, monoclonal antibodies).

General processes of the immune

response
Lymphocytes:
small
and
mononuclear cells primarily
associated with the immune
processes.
T lymphocytes: cells produced in
the thymus & responsible for
cell-mediated immunity.
B lymphocytes: cells produced in
the bone marrow & responsible
for antibody production.
Humoral immunity: mediated by
antibodies.
Cell-mediated immunity: cells
are directly involved in the
protective processes.

Kinetics of immune response

Primary
response:
Type of
reaction when
an animal
encounters an
antigen for the
first time

Secondary
response:
Type of
reaction when
an animal
encounters an
antigen more
than once

Antigen recognition and lymphocyte

stimulation
1. Antigen is taken up
by macrophages
upon entering the
tissues.
2. After modification, is
exhibited on the outer
membrane of the
cells.
3. Recognized by
lymphocytes via
antigen-specific
receptors carried on
their membranes.
4. T helper cells (Th
cells) recognize the
antigen and stimulate
the specific B, Tc or
Td cells.

Structure and antibody roles

Structure and antibody roles

Structure and antibody roles

Structure and antibody roles

Antigen-antibody reactions

Similar to the binding of an enzyme to its substrate.

Involves hydrophobic and electrostatic interactions.
Involves no subsequent chemical reaction and stability
depends upon the complementary shape of the antigen
and the binding site of the antibody.
Reversible and dissociate dependent upon the strength of
binding:
The strength of the binding of antibody to an antigen is
referred to its affinity and is defined by the equilibrium
constant K:

Production of antibody

Polyclonal antibodies: produce by immunization of an

animal involves injection of the pure antigen to stimulate
the immune system to produce antibodies. The antiserum
contains a heterogeneous mix of antibodies directed
against an antigen.
Monoclonal antibodies: produce by immunization of an
animal in traditional manner, but instead of allowing the
immune system to produce antibodies, the lymphocytes
are separated and fused in vitro with myeloma cancer
cells growing in cell culture. Contains homogeneous
serum containing only one antibody directed against an
antigen.

Production of monoclonal antibodies

Effects antigen-antibody complex

formation

Immunological methods of analysis

Analytical techniques-Precipitation
reactions

Many qualitative and quantitative methods for soluble antigens are

based on their precipitation by antibodies.
Immunoprecipitation in solution: the reaction is carried out in
an excess of antibody and a calibration is prepared by measuring the
turbidity of series of standard solutions of antigen. The formation of
antigen-antibody complexes are observed by measuring the
apparent absorption of light when incident light is scattered by the
complexes or by direct measurement of the scattered light.
Immunoprecipitation in gels: gels are used to stabilize the
precipitate, enabling both position and the area of the precipitate to
be measured. Antigen is permitted to diffuse into a gel that contains
a uniform conc. of antibody and precipitate will form at some point
in the conc. gradient of the antigen. The distance between the
precipitate and the original starting point of the antigen will be
proportional to its initial concentration. Forms the basis of single
radial immunodiffusion (SRID) technique (Procedure 7.1).

Analytical techniques-Immunoassay

Two broad approaches: competitive and non-competitive

immunoassays.
Competitive immunoassay: relies on the competition
between labelled and unlabelled antigens for a fixed and
limited number of antibody-combining sites.

Non-competitive or immunometric immunoassay:

employs antibodies in excess and for which there is no
competition for binding sites.

Components of immunoassay systems

The antibody: both polyclonal and monoclonal antibodies

have been used in immunoassays. It is essential to determine
the required optimum antibody concentration (the dilution
of antiserum (or titre) which will bind 50% of the labelled
antigen in the absence of unlabelled antigen) when using
competitive assay. Once the titre of an antiserum is known,
its specificity (or ability of an antiserum to recognize only
the antigen for which it was generated) may be assessed.
The label: the antibody or antigen needs to be labelled with
an appropriate molecule for detection. The molecule should
retain high signal efficiency and should have no effect on
their subsequent immunoreactivity (e.g: refer to Table 7.3)

Components of immunoassay systems

The choice of labels

1. Radioisotopes:
Carbon-14, hydrogen-3 (tritium) and iodine-125 are
commonly used radioisotopes.
Carbon-14 and hydrogen-3 are beta-emitting isotopes,
while iodine-125 emits both beta particles and gamma
radiation.
Carbon-14 and hydrogen-3 are example of internal labels
since the radioactive atom replaces an existing atom within
the antigen, while iodine-125 as external label because it is
necessary to attach the iodine covalently to the antigen.
Advantage of carbon-14 and hydrogen-3: labelled form of
molecules
is
identical
to
unlabelled
antigen,
immunoreactivity is maintained.

The choice of labels

1. Radioisotopes:
Disadvantage of carbon-14 & hydrogen-3: the efficiency of
the measurement of beta emission is less compared to
gamma emission of iodine-125.
Disadvantage of iodine-125: the covalent attachment of the
isotope to the antigen cause significant structural difference
between labelled and unlabelled antigen-loss of
immunoreactivity, but due to higher signal measurement, this
isotope is prefer to be used for immunoassay system.
2. Enzymes:
Are usually associated with solid-phase antibodies technique
known as enzyme-linked immunosorbent assay (ELISA).
The two-site assay employing two monoclonal antibodies is
the best technique of ELISA (refer to Figure 7.12).

The choice of labels

2. Enzymes:
The enzymes commonly used as labels include alkaline
phosphatase, horseradish peroxidase and beta-galactosidase.
The enzymes should be capable of being covalently linked to
the antigen or antibody without loss of their catalytic activity
or immunoreactivity.
The choice of enzyme is governed by the ability of substrate
and type of detector.
Enzymes also can be used to amplify the signal from the label
using cycling systems.
3. Luminescent labels:
Different types of luminescence depend on the source of
energy used to excite the molecules to higher energy state.
Radioluminescence immunoassay: occurs when energy is
supplied from higher energy particles. Antigen is labelled with
radioisotopes.

The choice of labels

3. Luminescent labels:
Chemiluminescence immunoassay: energy is derived fro a
chemical reaction. Antigen is tagged with a molecule such as
luminol or acridium ester that emits light with high quantum
yield on oxidation.
Bioluminescence immunoassay: excitation is performed by
biological molecule such as enzyme. Antigen is labelled with
luciferin which emits light when oxidized by luciferase
enzyme.
Fluorescence/ Photoluminescence immunoassay: excitation is
derived from light energy.
Antigen is labelled with
fluorescein or rhodamine.