Antibodies themselves: Human immunoglobulin G,
which contains human antibodies, is immunogenic in
experimental animals, because it is foreign to them.
Polysaccharides: Simple ones are not good. Longer ones,
especially if they are complex and/or associated with
Blood Group Antigens: A, B, AB and O.
LPS or Lipopolysaccharides: From cell wall of gramnegative bacteria.
Lipids are generally poor antigens.
Nucleic acids are generally poor antigens.
A class of proteins that migrate in the gamma fraction.
They are classified on the basis of heavy chains.
¾ IgG — Eighty percent plasma immunoglobulin, present
in all body fluids, transplacental,
¾ IgM — large molecule, pentameric in structure, present
in vascular system, activates complement
¾ IgA — present in body secretion, respiratory and GI tract
¾ IgE — involved in hypersensitivity and allergic reactions
¾ IgD — present in B cell surfaces.
What is the Structure of Antibody?
Basic model consists of 4 polypeptide chains
Heavy chains are structurally different for different
class of antibodies (Fig. 22.2).
What is the Kinetics of Antigen–Antibody Reaction?
The reaction complies with the law of mass action.
The higher the K, the stronger the reaction. The forces
What are the different ways of detection of antigen–
¾ Immunohistochemical techniques
¾ A clinical laboratory performs different kinds of tests
for detection of antigen–antibody reactions;
¾ Agglutination blood grouping, Widal test
¾ Latex agglutination—CRP, RF test
¾ Flocculation—VDRL test for syphilis
¾ Electrophoresis—protein biochemistry
¾ Chromatography—pregnancy tests.
How is Binder-ligand Assays Classified?
• Isotopic assays—radioimmunoassays
• Non-isotopic assays—enzyme Immunoassays, fluorescence polarization immunoassays.
What is the Difference Between All these Reactions?
All are basically antigen-antibody reaction.
The indicator used will differentiate the technology
What form of Reaction Takes Place in HLA Typing?
It is also antibody reaction in which the end product is
visualized by using a dye in a phase contrast microscope.
The reaction can also be visualized using fluorescent dyes
What is the Principle of HLA Typing?
It is called ad mixed lymphocytotoxicity test (MLT). In
this the antibody (antisera) is coated in the microwell. The
patient’s B or T lymphocytes containing HLA antigens is
added and incubated. Complement proteins are added
which will destroy the complex, if they are formed. The
dead and viable cells are differentiated and graded using
The principle is same for both cross-matching and
What are the Different Indicators Used in
Chemiluminescent dyes Acridinium ester CLIA
Chromogen Colloidal gold Chromatography
Microparticles Latex Latex agglutination
Despite advances in the design of immunoassays, the
problems of unwanted interference have yet to be
completely overcome. An ideal immunoassay should have
¾ The immunochemical reaction behavior should
be identical and uniform for both the reference
preparation and the analyte in the sample
¾ The immunochemical reaction of the antibody reagent
is uniform from batch to batch
¾ The immunochemical method is well standardized to
ensure that the size of measurement signal is caused
only by the antigen-antibody product
¾ For macromolecules the results declared in arbitrary
units (IU – International Units), the conversion to (SI)
units is not constant and depend on many factors.
Interference may be defined as “ the effect of a substance
present in an analytical system which causes a deviation of
the measured value from the true value, usually expressed
as concentration or activity.”
IFCC (International Federation of Clinical Chemistry)
offers the following d efinition – “Analytical interference is
the systematic error of measurement caused by a sample
component, which does not, by itself, produce a signal in
Assay interference can be “Analyte dependent or
It can increase or decrease the measured result.
Increase (positive interference) is due to lack of specificity.
Decrease (negative interference) is due to lack of
All factors associated with the constituents of the sample
are termed as preanalytical variables. They can be of two
Patient based: Such as incorrect sampling times and
environmental factors such as smoking, etc. may change
analyte concentration and consequently interpretation.
Specimen based: There are many factors that constitute
¾ Nature of the sample: For all immunoassays, serum
is the matrix of choice. Samples collected into tubes
containing sodium fluoride may be unsuitable for
some enzymatic immunoassay methods; preservation
with sodium fluoride may affect results. Impurities in
tracers interfere with direct dialysis methods for free
¾ Hemolysis and hyperbilirubinemia
¾ Lipemia — may cause interference with assays for fat
soluble compounds such as steroids
A fundamental problem with the analysis of components in
biological materials is the effect of the extremely complex
and variable mixture of proteins, carbohydrates, lipids,
and small molecules and salts constituting the sample.
The effect of these compounds on analytical techniques is
It can be defined as “ the sum of the effects of all the
components, qualitative or quantitative, in a system with
the exception of the analyte to be measured”.
Assay buffers: The ionic strength and pH of buffers
are vitally important, particularly in the case of
monoclonal antibodies with pI values of 5–9. The use of
binding displacers (blockers) may change the binding
characteristics of antibodies, particularly those of low
affinity. Detergents used in the buffers may contain
peroxides, which inhibit antigen-antibody reaction.
Immunoassay labels: Labels have a profound effect
on assays. The structure of most molecules, especially
haptens, may be dramatically changed by labeling, e.g.
by attachment of a radioactive iodine atom to a steroid.
Labeling antibodies with enzymes is less of a problem
Separation of the antibody-bound and free fractions: The
proportion of free analyte in the bound fraction and vice
versa is known as the “misclassification error”. Antibody
bound fraction may be efficiently separated from the free
analyte using solid-phase systems in which the antibody
is covalently linked to an inert support, e.g. the reaction
tube, a polystyrene bead, a cellulose or nylon.
Interfering proteins of general relevance include.
Albumin: May interfere as a result of its comparatively
huge concentration and its ability to bind as well as to
release large quantities of ligands.
Rheumatoid factors: These are autoantibodies usually
IgM class, and directed against the Fc portion of IgG. They
are not specific to rheumatoid arthritis and are found in
other autoimmune diseases, including systemic lupus
erythematosus, scleroderma and chronic active hepatitis.
Complement: These proteins bind to the Fc fragment of
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