Immunochemical Techniques: DR Amrita Bhowmik, PHD
Immunochemical Techniques: DR Amrita Bhowmik, PHD
Immunochemical Techniques: DR Amrita Bhowmik, PHD
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
A concentration gradient is established for only a single reactant and usually depends
on diffusion of an Antigen into agar impregnated with Ab.
• The antigen is applied into wells that are cut in the agarose gel containing dispersed
corresponding monospecific antibody.
• agarose plate is incubated at room temperature for 48-72 hours depending on the
specific protein in question.
• The antigen from the sample diffuses out from the wells into the agarose where the
antibody concentration is constant. In a distance from the well where antigen
concentration is equivalent to the antibody concentration (i.e., zone of equivalence
is reached in terms of the precipitin curve),
• the complex antigen-antibody precipitates and appears as a strong white ring
around the well.
• The square of the ring diameter is directly proportional to the antigen concentration.
This technique has been, however, currently largely replaced with
immunoturbidimetry or nephelometry
Double immunodiffusion/ Ouchterlony technique:
In double immunodiffusion reaction, the antigen and the antibody diffuse towards each other.
Reaction occurs three ways-
• a reaction of identity –It is based on wells punched into the agarose gel that are filled with
antigen or antibody solutions, respectively. Both antigen and antibody molecules are
allowed to diffuse radially into the gel surrounding the wells; and where the antigen and
specific reactive antibody meet, a precipitin line forms.
• a reaction of non-identity –If antiserum (antiserum, blood serum that contains specific antibodies against an
infective organism or poisonous substance) to several possible antigens is placed into the central well and
the outer wells are filled by different antigens, the precipitin bands form lines that intersect.
• a reaction of partial identity – if two antigenic mixtures are applied into two adjacent wells,
it is characterized by a formation of a spur. The spur means that the second antigen lacks an
epitope present in the first antigen that is recognized by one of the antibodies in the
antiserum.
2. Immunoelectrophoresis (IEP)
Immunoelectrophoresis is a qualitative method that combines protein
electrophoresis with immunodiffusion.
It is performed in two steps:
• The first one involves the separation of antigens according to their
charges/size in an electrical field.
• In the second step, a suitable antiserum (polyspecific or
monospecific) is applied to grooves running parallel to the
electrophoresis migration zone. The separated antigens and
antibodies are allowed to diffuse into the gel towards one another.
The precipitation line is formed in the area when the antigen with the
reacting antibody meets (Fig. 6).
Eg-, evaluation of human myeloma proteins.
Crossed Counter immunoelectrophoresis Immunofixation
immunoelectrophoresis (CIE) (IF)
(CRIE)
Two dimensional Two parallel lines of wells are First dimensional
punched in the agar electrophoresis is performed
in gel to separate the proteins
in the mixture
Electrophoresis is used in One row is filled with Ag solution Antiserum (Ab) spread
the 2nd dimension to derive and the opposing row is filled with directly on the gel causes the
the Ag into a gel Ab solution, proteins of interest to
that contains Ab specific Voltage is applied across the gel so precipitate which trapped
for the Ag of interest that the Ag and Ab move toward within gel matrix and
the anode, and Ab moves in the
opposite direction as a result of nonprecipitate proteins are
electrophoresis. removed by washing gel.
A precipitin line is formed where
they meet. The gel may be stained for
protein identification
More sensitive Provided within 1 -2 hrs
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