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Amrita Bhowmik
BSc & MSc, Biochemistry and Molecular Biology, JU
PhD Fellow, Biochemistry and Molecular Biology, DU
Lecturer
Dept of Applied Laboratory sciences
Bangladesh University of Health Sciences
Basic concepts and definitions
Lymphocytes occurs 2 major types-
• B cells (develop in the bone marrow, and
produce antibody)
• T cells (derived from bone marrow, then
develop in the thymus, generate cytokines and
kill virus or other pathogen)
They are responsible for specific recognition of
Antigens.
Antibody:
• Antibodies are produced by plasma cells that
result from differentiation of B lymphocytes
following stimulation with antigen.
• Antibodies are Immunoglobulins (Ig).
• They are capable of binding specifically to a wide
array of natural and synthetic antigens (proteins,
CHOs, nucleic acids, lipids and other molecules)
• There are 5 Types of Ig-IgA, IgG, IgM, IgD and IgE.
IgG
• IgG is the most prevalent immunochemical reagent in use.
• It is a Glycoprotein.
• Molecular weight (MW) 160 000.
• It is composed of two duplex chains with each set being
made up of a heavy (ɣ) and a light (α) chain joined by
disulfide bonds.
• Inter-chain disulfide bonds hold the duplex chains together
and create a symmetrical molecules.
• The variable amino acids sequence at the amino terminal
end if each chain determines the antigenic specificity of the
particular antibody.
• Each unique amino acid sequence is a product of a single
plasma line with a single specificity.
Schematic diagram of IgG antibody molecules
Antigen
• Definition: A substance that when introduced into the body stimulates the
production of an antibody. Antigens include toxins, bacteria, foreign blood
cells, and the cells of transplanted organs.
• In immunology, an antigen, or antibody generator is any substance which
provokes an adaptive immune response. An antigen is often foreign or
toxic to the body (for example, a bacterium) which, once in the body,
attracts and is bound to a respective and specific antibody. That is to say, an
antigen is molecule that also induces an immune response in the body.
Following are some general properties requisite for immunogenicity:
• Ag has area of structural stability within the molecule
• Structure is random
• Structure is foreign
• Has minimal MW of 4000-5000
• Is able to be metabolized
• A particular immunogenic configuration is accessible to the antibody-
forming mechanism
Epitope:
• Each unique region of the
molecular antigen that binds
a complementary antibody is
termed an Epitope.
• It is also called Antigenic
Determinant.
Paratope
The paratope is the part of an antibody which recognizes
an antigen, the antigen-binding site of an antibody. It is a
small region (of 15–22 amino acids) of the antibody's Fv
region and contains parts of the antibody's heavy and light
chains.[1]
*1. Ref
Monoclonal Ab:
• Monoclonal antibodies are products of a single clone of plasma
cells derived from B-lymphocytes.
• The unique ability of a monoclonal Ab to react with a single
epitope on a multivalent antigen.
• The majority of monoclonal Ab do not cross-link and precipitate
macromolecular Ag. For this reason monoclonal antibodies have
not found broad applicability in the clinical laboratory.
• They typically display excellent specificity, but poor ability to
precipitate antigen.
Polyclonal Ab:
A complex antigen is capable of eliciting a
multiplicity of antibodies with different
specificities that are derived from different cell
lines. Antibodies derived in this manner are
termed Polyclonal and exhibit diverse
specificities in their reactivity with immunogen.
Immunogen
• Antigens are recognized by specific lymphocytes or by antibodies,
not every antigen can evoke an immune response. Those antigens
that are capable of inducing an immune response are called
immunogens.
• Immunogen is an immunogenic material, that is either a protein or a
substance, coupled to a carrier.
• It is usually a protein, that when introduced into a foreign host is
capable of inducing the formation of an antibody in the host.
• We can define an immunogen as a complete antigen which is
composed of the macromolecular carrier and epitopes
(determinants) that can induce immune response.
Haptan
• The Haptens are low-molecular-weight compounds that may be
bound by antibodies, but cannot elicit an immune response.
Consequently the haptens themselves are nonimmunogenic and
they cannot evoke an immune response until they bind with a
larger carrier immunogenic molecule. The hapten-carrier complex,
unlike free hapten, can act as an immunogen and can induce an
immune response .
• It is a chemically defined determinant, that when conjugated to an
immunogenic carrier, stimulates the synthesis of antibody specific
to the haptan.
The strength or energy of interaction between the
Ag & Ab is described by two terms:
Affinity:
A thermodynamic quantity, defining the energy of interaction of
a single Ab-combining site and its corresponding epitope on
the antigen.
Avidity:
The overall strength of binding of Ab and Ag, and includes the
sum of the binding affinities of all the individual combining
sites on the antibody.
Example:
• IgG has two affinity binding sites and IgM has 10 per Ab
molecules.
• Affinity is a property of the substance bound (Ag).
• Avidity is a property of the binder (Ab)
IgM
• Immunochemical reactions form the basis of a
diverse range of sensitive and specific clinical
assays.
• The principles of the methods most commonly
used in the laboratory are – (next slide)
Book Reference:
Tietz
Fundamentals of Clinical Chemistry
4th or higher Edition
Principles
1. In a Typical immunochemical analysis, an
antibody is used as a reagent to detect the
chemical substance (Antigen) of interest.
2. The specificity and the high affinity of
antibodies for specific antigens coupled with
the unique ability of antibodies to cross-link
antigens allows identification and quantitation of
specific substances by a variety of methods.
Ag-Ab binding
Reaction mechanism
Several forces act cooperatively to produce Ag-Ab
binding. Three major contributing forces are-
1. Electrostatic Vandar Waals – London dipole-
dipole interaction (The interaction of two atoms, molecules, or nuclei by
means of their electric or magnetic dipole moments).
Where k1 (the rate constant for forward reaction)>>>k2 (the rate constant for
reverse reaction),
n is the number of epitopes per molecules
a and b are the number of Ag and Ab molecules per complex.
↑MW of polymer=
↑degree of linearity of polymer (minimum branching)=
↑aqueous solubility of protien
Qualitative Methods
Precipitation methods in gel
Direct agglutination
• In a direct agglutination test, the antigen is an integral part of the cell surface
(red blood cells, bacteria).
• A suspension of particles is directly agglutinated by specific antibodies present
in the examined sample.
• This assay is frequently used in the hematology for the determination of blood
group or in the immunological diagnostics or detection of specific antibodies
directed against naturally occurring antigens on the surface of some microbes.
Indirect agglutination
• Indirect agglutination assay utilizes particles (latex, colloid
gold) with the antigens (RBC are used as carrier) that have
been passively attached to their surface.(fig 9)
• Instead of the antigens, particles can also be coated by
specific antibodies. This technique is called reverse
agglutination and can be utilized for the detection of soluble
antigens (for example C-reactive protein or human chorionic
gonadotrophin /hCG) (Fig. 10).
Quantitative Methods
Precipitation methods
Membrane-bound EIA (lateral flow) tests are commonly used for rapid detection of
hormones (hCG, LH), viruses (hepatitis B and C), bacteria (Streptococcus,
Chlamydia, Helicobacter pylori), bacterial toxins, parasites (malaria),
therapeutic and illicit drugs, as well as biomarkers such as troponin
(cardiovascular disease) or prostate specific antigen(PSA, prostate cancer).
A homogeneous enzyme immunoassay :
A. Enzyme multiplied immunoassay technique (EMIT)
• It does not require a separation of bound and free labelled antibodies or antigens. It
is simple to perform and has been used for estimation of drugs, hormones and
metabolites.
• Sample containing the estimated antigen is mixed with a known quantity of the same
antigen labelled with enzyme (conjugate); and limited amount of specific antibody is
added.
• The unlabelled antigen from the sample competes with the conjugate for the
antibody.(competitive immunoassay)
• Binding of antibody on the conjugate results in loss of enzyme activity due to
blocking the enzyme active site or change of its conformation. The more unlabelled
antigen is present in the solution, the less conjugate will bind to the antibody, and
more enzyme activity will be preserved in the solution.
• Therefore, the enzyme activity is proportional to the antigen concentration in the
sample.
The method can be used for whole blood, serum, or urine. Enzyme multiplied
immunoassays can be fully automated with a fast throughput of clinical samples
especially in laboratories specializing in monitoring therapeutic drugs like
cyclosporin in transplant recipients.
B. Cloned enzyme donor immunoassay (CEDIA)
• This assay was the first RIA designed and developed using genetic
engineering techniques.
• Two active fragments (Enzyme donor ED and Enzyme Acceptor
EA) spontaneously reassemble to form active enzyme even if the
enzyme donor is attached to an Ag.
• Binding of Ab to the enzyme donor, Ag conjugates inhibits
reassembly. And no active enzyme is formed.
• Thus competition between Ag and the enzyme donor-Ag conjugate
for a fixed amount of Ab in the presence of the enzyme acceptor
modulates the measured enzyme activity.
• High Concentration of Ag produce the least (less) inhibition of
enzyme activity; low concentrations produce the greater inhibition.
One Example of Labeled Immunochemical Assays
eg- EIA